CN111936633A

CN111936633A - Microbial isolation and detection

Info

Publication number: CN111936633A
Application number: CN201980024570.5A
Authority: CN
Inventors: 丹尼尔·洛克哈特; 保罗·杰伊; 詹姆斯·图尔纳; 安德鲁·罗杰斯; 马修·克劳; 威廉·缪伦
Original assignee: Momentum Bioscience Ltd
Current assignee: Momentum Bioscience Ltd
Priority date: 2018-04-03
Filing date: 2019-04-03
Publication date: 2020-11-13
Also published as: GB201805479D0; JP7476466B2; EP3775254A2; US20210024877A1; CA3094056A1; WO2019193332A2; WO2019193332A3; JP2021519591A; JP2024038078A

Abstract

A method for separating microorganisms from non-microbial cells in a sample containing non-microbial cells includes incubating the sample with particles to form particle-microorganism complexes, and then separating the particle-microorganism complexes from the non-microbial cells. These methods are used to detect the presence of microorganisms in a sample that also contains non-microbial cells. Specific reagents and combinations of reagents enhance the selective capture of microorganisms in a mixed sample. Corresponding compositions and kits are also provided.

Description

Microbial isolation and detection

Technical Field

The present invention relates generally to the field of separating microorganisms from non-microbial cells in a sample and methods of detecting the presence or absence of microorganisms in a sample. The method typically relies on measuring the microbial enzyme activity (if any) present in the sample, wherein the sample also comprises a non-microbial source of enzyme activity. The present invention relies on a microbial source that effectively isolates the enzyme activity. Thus, the methods of the invention enable the determination of the presence of microbial pathogens in samples, such as unpurified blood, blood cultures, and other bodily fluids. The invention also relates to a kit comprising reagents for carrying out said method.

Background

In many cases, it is important to measure the presence and levels of certain molecules that are associated with cell viability. For example, measuring ATP levels in mammalian cells can be used for growth analysis and toxicology purposes. Culture methods can be used to detect small numbers of bacteria, but this technique can take days to complete, especially when trying to detect small numbers of bacteria and to detect slower growing microorganisms.

The detection of adenylate kinase as an indicator of viability has also been proposed (Squirell DJ, Murphy MJ, Leslie RL, Green JCD: A complexity of ATP and adenylate kinase as bacterial cell markers: correlation with agar plate counts)). WO 96/002665 describes a method for determining the presence and/or amount of microorganisms and/or their intracellular material in a sample, characterized in that the amount of adenylate kinase in the sample is assessed by the following procedure: mixing adenylate kinase in a sample with Adenosine Diphosphate (ADP), determining the amount of Adenosine Triphosphate (ATP) produced by the sample from the ADP, and correlating the amount of ATP so produced with the presence/amount of adenylate kinase and with the microorganism and/or its intracellular material, wherein the conversion of ADP to ATP is carried out in the presence of magnesium ions at a molar concentration sufficient to maximise the conversion of ADP to ATP.

In WO2009/007719, NAD-dependent ligase is described as a useful indicator of the presence of microorganisms in a sample. Ligases are enzymes that catalyze the ligation of nucleic acid molecules. Depending on the ligase involved, the ligation reaction requires ATP or NAD + as a cofactor.

WO2011/130584 describes a live microbial detection method based on DNA or RNA polymerase detection, wherein a sample is contacted with a nucleic acid substrate serving as a substrate for microbial polymerase, incubated under conditions suitable for polymerase activity from the intact microorganism and any resulting nucleic acid products are determined using a nucleic acid amplification technique such as quantitative polymerase chain reaction. Such assays are referred to as "ETGA assays", where ETGA stands for enzymatic template generation and amplification. For live microorganisms in crude samples, the problem with ETGA assays is the presence of contaminating polymerase activity outside the microorganism, which is derived from host (e.g., human) cells and dead microorganisms. The ETGA assay cannot distinguish microbial polymerase activity from host or dead microbial activity.

WO2010/119270 describes a method for removing DNA ligase activity outside of an intact microorganism.

WO2011/070507 describes the selective lysis of animal cells using non-ionic detergents and buffers.

WO/2017/182775 describes a method of detecting the presence of a microorganism in a sample which may also contain non-microbial cells, the method comprising selectively lysing the non-microbial cells, filtering the lysate and detecting the presence of a microorganism retained in or on the filter.

It is also known to use magnetic beads coated with specific binding moieties such as antibodies. The specificity of these products is defined by the specificity of antibodies or other binding ligands, which are generally selected for a specific purpose with a high degree of specificity to allow the isolation of specific microorganisms.

WO03/102184 describes methods, compositions and kits for concentrating or isolating cells (e.g. bacteria) to form complexes with the cells to aggregate them using flocculants such as polyamines or cationic detergents. The separation of aggregated cells can be achieved with a solid phase capable of binding cells, such as magnetic beads.

WO01/53525 describes a method of isolating cells (e.g.microorganisms) from a sample, which method comprises binding the cells to a solid support via a carbohydrate ligand immobilised on the solid support. Kits for carrying out this method are sold by Diasorin Molecular ("Bugs' n Beads^TM"kit").

Other kits for isolating microorganisms include magnetic beads of ApoH-Technologies Peps 6. The beads are coated with the synthetic molecule Peps6, which is derived from the apolipoprotein H protein (ApoH), also known as beta-2 glycoprotein.

Disclosure of Invention

The present inventors have realised that in samples taken from subjects suspected of carrying microbial infections there are more levels of nucleated blood cells (leukocytes) than previously thought, although most samples are not actually from infected subjects. This has led to a need for improved methods of isolating potential microorganisms from blood cells, particularly leukocytes, in blood samples taken from patients screened for infection. The present invention relates to the separation of microorganisms from non-microbial cells in a sample by selectively capturing microorganisms having particles (e.g., magnetic particles) that form particle-microorganism complexes and separating the particle-microorganism complexes from the non-microbial cells (e.g., using a magnetic field). This provides a solution to the above problems, thereby enabling the detection of the presence or absence of microorganisms in a sample. The inventors have surprisingly found that such separation can be achieved with particles (e.g. magnetic particles) that are not coated with a ligand. The inventors have found that certain agents are particularly useful in methods that ensure good separation of microorganisms from non-microbial cells, particularly in complex samples such as blood, milk and urine.

The present invention provides a method of separating microorganisms from non-microbial cells in a sample containing non-microbial cells, the method comprising: a) incubating a sample with a particle having an outer surface to form a particle-microorganism complex; and b) separating the particle-microorganism complex from the non-microbial cell.

The present invention provides a method of separating microorganisms from non-microbial cells in a sample containing non-microbial cells, the method comprising: a) incubating the sample with a particle to form a particle-microorganism complex, wherein the particle has an outer polymer surface; and b) separating the particle-microorganism complex from the non-microbial cell.

The present invention provides a method of separating microorganisms from non-microbial cells in a sample containing non-microbial cells, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the incubating step is performed in the presence of sodium polyanetholesulfonate and/or an agent that selectively lyses non-microbial cells in the sample while leaving microorganisms present in the sample intact; and b) separating the particle-microorganism complex from the non-microbial cell.

The present invention provides a method of separating microorganisms from non-microbial cells in a sample containing non-microbial cells, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the incubating step is performed in the presence of sodium polyanetholesulfonate and/or a detergent; and b) separating the particle-microorganism complex from the non-microbial cell.

The present invention provides a method of separating microorganisms from non-microbial cells in a sample containing non-microbial cells, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes; and b) separating the particle-microorganism complex from the non-microbial cell; wherein the particles have an outer surface that is not coated with any one of: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin protein.

In this method, the incubating step can be performed in the presence of polyanetholesulfonic acid sodium and/or an agent that selectively lyses non-microbial cells in the sample while leaving microorganisms present in the sample intact.

In this method, the incubating step can be performed in the presence of sodium polyanetholesulfonate and/or a detergent. Detergents are examples of reagents that selectively lyse non-microbial cells in a sample while leaving intact the microorganisms present in the sample.

In the method, the method may further comprise washing the isolated particle-microorganism complex to remove non-microbial cells or lysate; optionally wherein the isolated particle-microorganism complex is washed with a solution comprising a detergent and/or sodium chloride.

The reagent that selectively lyses non-microbial cells in the sample while leaving intact the microorganisms present in the sample may comprise a combination of a detergent and one or more enzymes. The one or more enzymes may comprise a protease and/or a dnase. Suitable detergents and enzymes are discussed herein.

In this process, step b) may be carried out by any suitable separation means. For example, separation can be achieved using a magnetic field to attract the particle-microorganism complexes or centrifugation.

In the method, step b) may further comprise removing non-microbial cells from the particle-microbe complex.

In this method, the non-microbial cells in the sample may be selectively lysed prior to step a) while leaving intact the microorganisms present in the sample.

In this method, selectively lysing non-microbial cells in the sample while leaving intact any microbes present in the sample can include freezing and thawing the sample.

In this method, selectively lysing non-microbial cells in the sample while leaving intact any microorganisms present in the sample may comprise adding a detergent.

In this method, step a) may be carried out in the presence of a buffer. The buffer may have a pH between 7.4 and 8.5.

In this process, step a) may be carried out in the presence of sodium chloride. Sodium chloride may be present at a concentration between 50 and 500 mM. Preferably, sodium chloride may be present at a concentration of about 150 mM.

In this method, the reagent that selectively lyses non-microbial cells in the sample while leaving intact the microorganisms present in the sample may be a detergent. In this method, the detergent may be non-ionic. In this method, the detergent may not be conjugated to a particle capable of forming a complex with the microorganism. Thus, typically the detergent forms part of the solution to which the particles are added, and not of the particles themselves.

In the method, the particles may have a diameter between 0.1 and 3 μm or between 0.1 and 2 μm. Preferably, the particles have a diameter between 0.1 and 1.0 μm.

In this method, the particles may be (and typically are) magnetic. The particles may be superparamagnetic. The particles may comprise iron oxide. The iron oxide may include magnetite and/or maghemite. The iron oxide may not contain Fe in a 1:1, 2:1, 3:1, or 4:1 ratio²⁺And Fe³⁺。

The outer surface of the particle capable of forming a complex with a microorganism may comprise a polymer; optionally, the polymer may be carbon-based. The polymer may not comprise an inorganic polymer. The polymer may comprise polystyrene and/or poly (styrene/divinylbenzene).

In this method, the outer surface of the particle capable of forming a complex with a microorganism may comprise or be coated with any one or more of: i) a carboxylic acid group; ii) an amino group; iii) a hydrophobic group; and iv) streptavidin; optionally, a carboxylic acid group; ii) an amino group; iii) the hydrophobic group may not be part of the polypeptide.

In the method, the microorganism may be a pathogenic microorganism. For example, the pathogenic microorganism may be a pathogenic bacterium or fungus.

In the method, the non-microbial cells may comprise red blood cells and/or white blood cells.

In this method, the sample may comprise non-microbial cells at a concentration of 20,000 to 5 million cells per ml. The sample may comprise non-microbial cells at a concentration of at least about 100,000 cells per milliliter. Preferably, the sample may comprise non-microbial cells at a concentration of at least about 20,000 cells per ml.

The sample is a sample that contains or is suspected of containing a microorganism. The sample comprises non-microbial cells, which microorganisms may provide an unwanted background when aimed at detecting the presence of microorganisms in the sample and potentially identifying and/or quantifying the microorganisms present in the sample. Thus, in some embodiments, the sample may comprise blood, urine, saliva, or milk. The blood sample may be any sample containing blood cells. The blood sample may be or may comprise whole blood (e.g. blood broth).

The present invention provides a method of separating microorganisms from non-microbial cells in a sample containing non-microbial cells, the method comprising: (a) incubating the sample with particles (e.g., magnetic particles) to form particle-microorganism complexes; and (b) separating the particle-microorganism complex from the non-microbial cell (e.g., using a magnetic field).

In this method, the particles (e.g., magnetic particles) may have an outer polymer surface that is not coated with any one of the following: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin, (v) a polyamine or (vi) a cationic detergent.

In this method, the particles (e.g., magnetic particles) may have an outer surface (e.g., outer polymer surface) that is not coated with any one of the following: (i) an antibody, (ii) a carbohydrate, or (iii) an innate immune system protein.

In this method, the particles (e.g., magnetic particles) may have an outer surface (e.g., outer polymer surface) that is not coated with any one of the following: (i) an antibody, (ii) a carbohydrate, (iii) a peptide protein derived from apolipoprotein H, (iv) a mannose-binding lectin, or (v) a flocculant agent (e.g. as defined in WO 03/102184).

In this method, the particles (e.g., magnetic particles) may have an outer surface (e.g., outer polymer surface) that is not coated with any one of the following: (i) an antibody, (ii) a carbohydrate, (iii) an innate immune system protein or (iv) a flocculant (e.g. as defined in WO 03/102184).

In this method, the particles (e.g., magnetic particles) can have an outer surface (e.g., outer polymer surface) that is not coated with ligands.

In this method, the particles (e.g., magnetic particles) can have an outer surface (e.g., outer polymer surface) that is coated with streptavidin and not with ligands.

In this method, the particles (e.g., magnetic particles) can have an outer surface (e.g., outer polymer surface) that is coated with only streptavidin.

In this method, the particles (e.g., magnetic particles) may have an outer surface (e.g., outer polymer) surface that is coated with only carboxyl groups.

In this method, the particles (e.g., magnetic particles) may have an outer surface (e.g., an outer polymer surface) that is not coated with any molecules or moieties capable of binding microorganisms.

In this method, the particles (e.g., magnetic particles) may have an outer surface (e.g., outer polymer surface) that is not coated with any molecules or moieties.

By "coated" is meant attached to an outer surface (e.g., an outer polymeric surface). The skilled person will know means for attaching molecules and chemical groups to the outer surface (e.g. outer polymer surface) of a particle (e.g. a magnetic particle).

The isolation method of the present invention can be used to detect whether a microorganism is found in a sample that also contains non-microbial cells. Once the microorganisms are isolated from potential background signal sources, they can be specifically and sensitively detected using a variety of techniques. Accordingly, the present invention also provides a method of detecting the presence or absence of a microorganism in a sample, which sample may also comprise non-microbial cells, which method comprises: a) incubating the sample with particles to form particle-microorganism complexes; b) separating the particle-microorganism complex from the non-microbial cells; and c) detecting the presence or absence of a microorganism in the particle-microorganism complex.

The present invention also provides a method of detecting the presence or absence of a microorganism in a sample, which sample may also comprise non-microbial cells, which method comprises: a) incubating the sample with a particle to form a particle-microorganism complex, wherein the particle has an outer polymer surface; b) separating the particle-microorganism complex from the non-microbial cells; and c) detecting the presence or absence of a microorganism in the particle-microorganism complex.

In a related aspect, the invention provides a method of detecting the presence or absence of a microorganism in a sample, which sample may also comprise non-microbial cells, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the incubating step is performed in the presence of sodium polyanetholesulfonate and/or an agent that selectively lyses non-microbial cells in the sample while leaving microorganisms present in the sample intact; b) separating the particle-microorganism complex from the non-microbial cells; and c) detecting the presence or absence of a microorganism in the particle-microorganism complex.

Similarly, the present invention provides a method of detecting the presence or absence of a microorganism in a sample, which sample may also comprise non-microbial cells, which method comprises: a) incubating the sample with particles to form particle-microorganism complexes, wherein the incubating step is performed in the presence of sodium polyanetholesulfonate and/or a detergent; b) separating the particle-microorganism complex from the non-microbial cells; and c) detecting the presence or absence of a microorganism in the particle-microorganism complex.

The present invention further provides a method of detecting the presence or absence of a microorganism in a sample, which sample may also comprise non-microbial cells, which method comprises: a) incubating the sample with particles to form particle-microorganism complexes; b) separating the particle-microorganism complex from the non-microbial cells; and c) detecting the presence or absence of a microorganism in the particle-microorganism complex; wherein the particles have an outer surface that is not coated with any one of: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin protein.

In this method, the incubating step can be performed in the presence of polyanetholesulfonic acid sodium and/or an agent that selectively lyses non-microbial cells in the sample while leaving microorganisms present in the sample intact. In this method, step c) may comprise (i) detecting the enzymatic activity of a nucleic acid molecule associated with the microorganism; (ii) direct detection of microorganisms by cell counting or microscopy; (iii) detecting microorganisms after cell culture; (iv) (iv) detecting the microorganism by PCR, or (v) detecting the microorganism by nucleic acid sequencing.

In the method, step c) may include the steps of: i) lysing the microorganisms in the particle-microorganism complex; ii) incubating the lysate with a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of the microorganism; and iii) specifically determining the presence or absence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying enzyme on the substrate nucleic acid molecule to indicate the presence or absence of a microorganism. In the method, step (i) may comprise adding a lysis reagent comprising a substrate nucleic acid molecule. In this method, the nucleic acid modifying enzyme may comprise a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I.

Since microorganisms are a common source of infection in a subject, the methods of the invention can be used to identify infections caused by microorganisms. Accordingly, the invention also provides a method of detecting the presence or absence of a microbial infection in a subject, the method comprising performing any of the methods described herein on a sample from the subject (detecting a microorganism in the sample).

The method may further comprise washing the isolated particle-microorganism complex to remove non-microbial cells or lysate.

In the method, step (b) may further comprise removing non-microbial cells from the particle-microbe complex.

In this method, the sample may comprise non-microbial cells at a concentration of 20,000 to 5 million cells per ml. The sample may comprise non-microbial cells at a concentration of at least about 100,000 cells per ml. Preferably, the sample may comprise non-microbial cells at a concentration of at least about 20,000 cells per ml.

The sample is a sample that contains or is suspected of containing a microorganism. The sample comprises non-microbial cells, which microorganisms may provide an unwanted background when aimed at detecting the presence or absence of microorganisms in the sample and potentially identifying and/or quantifying the microorganisms present in the sample. Thus, in some embodiments, the sample may comprise blood, urine, saliva, or milk. The blood sample may be any sample containing blood cells. The blood sample may be or may comprise whole blood (e.g., blood culture).

The present invention provides a method of detecting the presence or absence of a microorganism in a sample, which sample may also comprise non-microbial cells, which method comprises: (a) incubating the sample with particles (e.g., magnetic particles) to form particle-microorganism complexes; (b) separating the particle-microorganism complex from the non-microbial cell (e.g., using a magnetic field); and (c) detecting the presence or absence of a microorganism in the particle-microorganism complex.

In this method, the particles (e.g., magnetic particles) may have an outer surface (e.g., outer polymer surface) that is not coated with any one of the following: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from apolipoprotein H, (iv) a mannose-binding lectin protein, (v) a polyamine or (vi) a cationic detergent.

In this method, the particles (e.g., magnetic particles) may have an outer polymer surface that is not coated with any one of the following: (i) an antibody, (ii) a carbohydrate, or (iii) an innate immune system protein.

In this method, the particles (e.g., magnetic particles) may have an outer polymer surface that is not coated with any one of the following: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin, or (v) a flocculant agent (e.g. as defined in WO 03/102184).

In this method, the particles (e.g., magnetic particles) may have an outer surface (e.g., outer polymer surface) that is coated with only carboxyl groups.

In this method, step (c) may comprise (i) detecting the enzymatic activity of a nucleic acid molecule associated with the microorganism, (ii) detecting the microorganism directly by cell counting or microscopy, or (iii) detecting the microorganism after cell culture.

The detection of the presence or absence of microorganisms in the particle-microorganism complex according to all relevant aspects of the invention may be carried out according to any desired method. The method may comprise simply detecting the presence or absence of one or more microorganisms. If present, it may relate to the quantification of the microorganism. In some embodiments, it may also relate to the characterization of microbial properties. Thus, detection of bacteria and/or fungi can be performed. Gram positive bacteria can also be distinguished from gram negative bacteria. Identification of organisms and antimicrobial susceptibility may also be performed.

After removal (or recovery) of the microorganisms from the particle-microorganism complex, detection can be performed. The recovered microorganisms may be lysed prior to detection. The intact microorganism or lysate can be recovered after lysis of the microorganism (as discussed in further detail herein).

Preferably, the detection is performed without prior removal (or recovery) of the microorganisms from the particle-microorganism complex. This embodiment is particularly useful in applying the present invention to a magnetic bead processing instrument.

The detection of the presence or absence of a microorganism can include detection of an enzymatic activity or a nucleic acid molecule associated with the microorganism. Direct detection of microorganisms by cell counting or microscopy; or detecting the microorganism after cell culture.

Detection of nucleic acid molecules associated with microorganisms is known in the art and can be performed at the DNA or RNA level. This may be done by any suitable method, such as amplification (e.g. PCR) or sequencing (especially next generation sequencing). Such methods can utilize sequence differences between microorganisms and non-microorganisms (e.g., human, DNA and RNA). Such methods may include lysing the microorganism (e.g., in the form of a particle-microorganism complex) to release the nucleic acid component.

Direct detection of microorganisms is also known. This may involve cellular analysis, for example by flow cytometry. It may involve the use of microscopy, for example to visualize the microorganisms recovered from the particle-microorganism complex or to visualize the microorganisms in the particle-microorganism complex.

In order to expand the number of microorganisms, detection of microorganisms may also be performed after cell culture. Thus, the microorganisms initially captured in the particle-microorganism complex may be incubated for a set time prior to detection. The culture method can directly detect the microorganisms in the original sample.

However, in some preferred embodiments, detecting the presence or absence of a microorganism can include detecting an enzyme activity associated with the microorganism. Suitable enzymatic activities are typically nucleic acid modifying activities and are discussed in more detail herein.

Thus, in the method, step (c) may comprise the steps of: (i) lysing the microorganisms in the particle-microorganism complex; (ii) incubating the lysate with a nucleic acid molecule that serves as a substrate for nucleic acid modifying activity of the microorganism; (iii) the presence or absence of a modified nucleic acid molecule resulting from the action of a nucleic acid modifying enzyme on the substrate nucleic acid molecule is specifically determined to indicate the presence or absence of a microorganism.

In the method, step (i) may comprise adding a lysis reagent comprising a substrate nucleic acid molecule.

Incubating the sample refers to contacting the sample with the particle under conditions that favor the formation of particle-microorganism complexes. In some embodiments, the step of incubating the sample with particles (e.g., magnetic particles) comprises contacting the particles (e.g., magnetic particles) with the sample at a specified temperature (e.g., 37 ℃) for a fixed period of time (e.g., 30 min). Incubation can be performed with or without shaking (e.g., by a platform shaker, orbital shaker, or a shaking incubator set at 500-.

The lysis of the microorganisms in the particle-microorganism complex allows the detection of nucleic acid molecules or enzymes, such as nucleic acid modifying enzymes, within the microorganisms. Lysis may be achieved by adding a lysis mixture. Lysis mixtures are generally useful in the process of the invention. The lysis mixture may comprise a specific mixture of components to ensure efficient lysis of the microorganism without adversely affecting the nucleic acid molecules and/or the enzymatic activity, e.g. nucleic acid modifying activity, within the cell. The components may be selected from carriers/serum proteins such as BSA, surfactants/detergents, metal halide salts, buffers, chelating agents and the like. In its basic form, the lysis mixture of the invention may comprise the following components:

1. surfactant/detergent

2. Serum proteins, e.g. albumin (e.g. BSA)

3. Buffer solution

4. Nucleotides, e.g. dNTPs

5. Nucleic acid molecules (which serve as substrates in the assays of the invention).

Suitable lysis mixtures are listed below:

l1: 252mL in 360mL LM

1.46％(w/v)BSA

0.15％Triton X100

0.15％Tween 20

L2: 36mL in 360mL LM

100mM ammonium sulfate

20mM magnesium sulfate heptahydrate

100mM potassium chloride

200mM Tris-HCl[pH 8.0]

L3: 36mL in 360mL LM

0.1. mu.M PTO-AS oligonucleotide

0.1. mu.M PTO-S1 oligonucleotide

20mM Tris-HCl[pH 8.5]

10mM potassium chloride

10μM EDTA

10mM dNTP: 3.6mL in 360mL LM

PTO-IPC stock solution: 180 μ L in 360mL LM

H₂O: in 360mL LM-32.4 mL

"PTO-AS oligonucleotide" refers to an antisense oligonucleotide comprising a phosphorothioate nucleotide. "PTO-S1 oligonucleotide" refers to a sense oligonucleotide comprising a phosphorothioate nucleotide. The two oligonucleotides hybridize to each other to form a substrate nucleic acid molecule.

"PTO-IPC" refers to IPC molecules comprising phosphorothioate nucleotides.

Suitable substrates and IPC molecules are discussed in further detail herein.

Exemplary amounts and concentrations of each component are listed, but may be modified as would be readily understood by one skilled in the art.

Lysis may also require destruction of the cells. For example, the lysis mixture may be used in combination with physical and/or enzymatic means to disrupt the cells. However, in general, methods of lysing cells avoid the use of physical disruption. In some embodiments, the physical disruption employs a disruptor. Disruptors may bind beads, such as glass beads, to lyse cells. Suitable equipment is commercially available and includes the Disproptor Genie manufactured by Scientific Industries, Inc. Sonication may be used, for example the application of an ultrasonic horn. In some embodiments, enzymatic disruption may entail the use of one or more reagents selected from lysostaphin, lysozyme, and/or a lyase.

Once the microorganism (if present in the sample) is lysed, the released nucleic acids and/or enzymes can be detected to indicate whether the microorganism is present in the sample. In some embodiments, the lysate is incubated with a nucleic acid molecule that serves as a substrate for nucleic acid modification activity (of the microorganism). The presence or absence of a modified nucleic acid molecule resulting from the action of a nucleic acid modifying enzyme on the substrate nucleic acid molecule is then determined to indicate the presence or absence of a microorganism. Nucleic acid substrate molecules are designed according to the nucleic acid modification activity to be detected. The skilled person is well able to design suitable substrate nucleic acid molecules. Although the initial sample comprises a non-microbial source of nucleic acid modifying activity, the method of the invention prevents such contaminating activity from acting on the substrate nucleic acid molecules.

The nucleic acid modifying enzyme may comprise a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I.

The nucleic acid modifying enzyme may comprise a ligase, optionally wherein the nucleic acid modifying enzyme is an NAD-dependent ligase.

The invention further provides a method of detecting the presence or absence of a microbial infection in a subject, the method comprising performing a method of any of the methods described herein on a sample from the subject, optionally wherein the sample comprises blood from the subject.

The method may further comprise washing the isolated particle-microorganism complex to remove non-microbial cells or lysate. The washing step may remove inhibitors, such as PCR inhibitors, for subsequent analysis. The washing step may be performed under conditions that do not dissociate the particle-microorganism complexes.

Typical nucleic acid modification activities that can be detected according to the methods of the invention include polymerase and/or ligase activity. In certain embodiments, the nucleic acid modifying enzyme comprises a DNA or RNA polymerase. In some embodiments, the DNA polymerase comprises or is DNA polymerase I. In some embodiments, the nucleic acid modifying enzyme comprises a ligase. In certain embodiments, the nucleic acid modifying enzyme comprises or is an NAD-dependent or ATP-dependent ligase. NAD-dependent ligases are only found in (true) bacteria and, therefore, detection of such activity may provide other levels of specificity. Further discussed in W02009/007719 and WO2010/119270 (the relevant disclosures of which are incorporated herein). Alternatively, other nucleic acid modification activities associated with viability, such as phosphatase, kinase, and/or nuclease activity, can be measured.

In some embodiments, the effect of the nucleic acid modifying activity on the substrate nucleic acid molecule results in an extended nucleic acid molecule. This can be achieved by chain elongation (polymerase activity) and/or by ligation of two nucleic acid molecules (ligase activity). In some embodiments, a substrate is utilized that can be acted upon by a polymerase or ligase, as either activity is indicative of the presence of a microorganism in the sample. In some embodiments, the relevant activities can be distinguished based on the novel nucleic acid molecules produced.

The substrate may be a template for nucleic acid modifying activity of the microorganism. For example, the substrate may be a template for a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I.

Suitable nucleic acid molecules for use as substrates for nucleic acid modification activity of microorganisms are described in WO2011/130584, WO2010/119270 and WO2009/007719 (the relevant disclosures of which are incorporated herein by reference). In the case of phosphatase activity, suitable nucleic acid molecules are disclosed in WO2006/123154, the disclosure of which is incorporated herein by reference.

In some specific embodiments, the (substrate) nucleic acid molecule used in the method of the invention is at least partially double-stranded and comprises a uracil residue in the complementary strand, and in particular the step of determining the presence or absence of the modified nucleic acid molecule comprises adding Uracil DNA Glycosylase (UDG) to the sample to degrade uracil residues in the complementary strand.

In certain embodiments, the (substrate) nucleic acid molecule comprises DNA. In certain embodiments, the (substrate) nucleic acid molecule comprises DNA and is partially double-stranded.

In some embodiments, the (substrate) nucleic acid molecule comprises a nucleic acid consisting of a sense oligonucleotide (DNA) strand and an antisense oligonucleotide (DNA) strand, wherein the two strands overlap to form a double-stranded region and, in the presence of polymerase activity, the single-stranded portion of the antisense oligonucleotide strand serves as a template and the sense oligonucleotide strand of the double-stranded region serves as a primer to produce an extension product;

in certain embodiments, a first strand of a partially double-stranded (substrate) nucleic acid molecule comprises (or consists of) a synthetic nucleotide (e.g., a phosphorothioate nucleotide), while a second (complementary) strand comprises (or consists of) a uracil residue, and optionally, a synthetic nucleotide (e.g., a phosphorothioate nucleotide). Preferably, the double stranded region comprises the 3' terminal regions of the first and second (complementary) strands. Preferably, the second (complementary) strand comprises a base (e.g., dideoxycytidine) at its 3' end that blocks DNA polymerase-mediated elongation of the second strand. Such partially double-stranded (substrate) nucleic acid molecules are described, for example, in Zweitzig et al, 2012 (characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable bacteria. nucleic acid studies 40, 14, e109, 1-12). Preferably, the double-stranded region is at least 5, at least 10, at least 15, at least 20 or at least 25 nucleotides; optionally, the double-stranded region is no more than 50 nucleotides. As described herein, the first strand may be extended in the incubation step by polymerase activity of the microorganism in the sample using unprotected (or standard) dntps to form an extended first strand comprising unprotected (or standard) nucleotides. This step relies on the use of the second strand as a template (upstream of the region of complementarity between the first and second strands). After the incubation step, the second (complementary) strand may be degraded by adding Uracil DNA Glycosylase (UDG) to the sample, thereby rendering the extended first strand a single-stranded molecule comprising synthetic nucleotides and unprotected nucleotides. After degradation of the second strand, the extended first strand of the (substrate) nucleic acid molecule may be detected in an amplification step. The inventors have found that the use of partially double stranded (substrate) nucleic acid molecules as described above improves the detection of microorganisms in a sample.

In some embodiments, the substrate nucleic acid molecule is pre-modified so as to protect it from nuclease activity, i.e., the nucleic acid molecule is modified to protect it from nuclease activity prior to addition to the assay. The inventors have determined that protecting a substrate nucleic acid molecule from nuclease activity is advantageous in the assays of the invention. More specifically, incorporation of protected nucleic acid molecules into the methods of the invention improves the sensitivity of detection. To protect a nucleic acid molecule from nuclease activity, any suitable means may be employed. Non-limiting examples include incorporation of methylation into nucleic acid molecules, end modifications, such as protection of the 3 'and/or 5' ends, and incorporation of synthetic nucleotides. In particular embodiments, the synthetic nucleotide comprises a phosphorothioate nucleotide and/or a locked nucleic acid nucleotide. Preferably, the synthetic nucleotide is a phosphorothioate nucleotide. In certain embodiments, the synthetic nucleotide replaces at least one to all nucleotides in the nucleic acid molecule.

The (substrate) nucleic acid molecule may comprise any natural nucleic acid and natural or synthetic analogues capable of acting through a nucleic acid modifying activity to generate a (newly detectable) nucleic acid molecule. In particular embodiments, the substrate may be extended and/or linked. In some embodiments, a combination of nucleic acid substrate molecules can be used to allow detection of polymerase and ligase activities.

The nucleic acid substrate may be present in excess, and in particular in large molar excess, relative to the nucleic acid modifying activity (provided by the microorganism) in the sample. Since new extended or ligated nucleic acid molecules can be detected, the presence of such molecules in the sample is only necessary to carry out the detection method efficiently. Thus, the method of the invention is not detrimental if other nucleic acid molecules are present in the sample, e.g.from the microorganism to be detected or from a mammal or other source that may be found in the sample to be detected.

The inventors have previously investigated the use of Internal Positive Control (IPC) molecules in the context of their methods. Thus, according to all aspects, the invention may rely on the inclusion of IPC molecules. In some embodiments, the IPC is included in a substrate nucleic acid molecule, such that the IPC is exposed to the same conditions. In some embodiments, the IPC molecule is pre-modified to protect it from nuclease activity, i.e., it is modified to protect it from nuclease activity prior to addition of the nucleic acid molecule to the assay. The inventors have determined that it is advantageous to protect IPC molecules against nuclease activity in the context of the assays of the invention. To protect a nucleic acid molecule from nuclease activity, any suitable means may be employed. Non-limiting examples include incorporation of methylation into nucleic acid molecules, end modifications, such as protection of the 3 'and/or 5' ends, and incorporation of synthetic nucleotides. In particular embodiments, the synthetic nucleotide comprises a phosphorothioate nucleotide and/or a locked nucleic acid nucleotide. Preferably, the synthetic nucleotide is a phosphorothioate nucleotide. In certain embodiments, the synthetic nucleotides replace at least one to all nucleotides in the IPC molecule. Preferably, the substrate and IPC molecule are modified in the same way, as it is advantageous for them to behave similarly in the assay of the invention.

In some embodiments, an Internal Positive Control (IPC) nucleic acid molecule comprises the same primer binding site as the substrate nucleic acid molecule, such that primer binding is competed in a nucleic acid amplification reaction comprising both the nucleic acid molecule and the IPC.

In all methods of the invention, specifically determining whether a modified nucleic acid molecule is present may comprise, consist essentially of, or consist of: and (3) a nucleic acid amplification step. This serves to make the method of the invention most sensitive. Such amplification techniques are well known in the art and include techniques such as PCR, NASBA (Compton, 1991), 3SR (Fahy et al, 1991), rolling circle replication, Transcription Mediated Amplification (TMA), Strand Displacement Amplification (SDA) Clinical Chemistry 45: 777-784, 1999, DNA oligomer self-assembly procedures of US6261846 (incorporated herein by reference), Ligase Chain Reaction (LCR) (Barringer et al, 1990), selective amplification of target polynucleotide sequences (US 6410276), random priming PCR (WO 90/06995), consensus sequence priming PCR (US 4437975), invader technique, strand displacement technique and nick-displacement amplification (WO 2004/067726). The above list is not intended to be exhaustive. Any nucleic acid amplification technique may be used, provided that the appropriate nucleic acid product is specifically amplified.

Similarly, sequencing-based methods can be employed in some embodiments to include any range of next generation sequencing platforms, such as sequencing by synthetic clonal amplification (Illumina), pyrosequencing, 454 sequencing (Roche), Nanopore sequencing (e.g., Oxford Nanopore), ion torrent (ThermoFisher), and single molecule real-time (SMRT) sequencing (Pacific Biosystems). The fact that a novel nucleic acid molecule is generated means that sequencing methods can confirm the presence or absence of the modified nucleic acid molecule and also provide quantification of the molecule.

Amplification is achieved by using amplification primers specific for the sequence of the modified nucleic acid molecule to be detected. To provide specificity for a nucleic acid molecule, a primer binding site corresponding to an appropriate region of the sequence can be selected. The skilled reader will appreciate that nucleic acid molecules may also comprise sequences other than primer binding sites required for the detection of novel nucleic acid molecules resulting from modification activity in a sample, e.g. RNA polymerase binding sites or promoter sequences may be required for isothermal amplification techniques (e.g. NASBA, 3SR and TMA).

One or more primer binding sites may bridge the ligation/extension boundary of the substrate nucleic acid molecule such that only ligation/extension occurs to produce an amplification product. Alternatively, primers can bind to either side of the ligation/extension boundary and amplify directly across the boundary such that only the ligated/extended nucleic acid molecule is formed to (exponentially) produce an amplification product. Primers and substrate nucleic acid molecules can be designed to avoid non-specific amplification (e.g., genomic DNA in a sample).

The primer may suitably incorporate synthetic nucleotide analogues, or may be, for example, RNA or PNA based or a mixture thereof. Depending on the detection mode employed, the primers may be labeled, for example, with a fluorescent label and/or a FRET pair.

The probe may be used, or may be labeled as necessary. The detection method may require the use of nucleotide probes other than, or as a substitute for, primers. For example, a branched DNA assay that does not require the use of primers may be employed in some embodiments.

In certain aspects, the methods of the invention are performed using nucleic acid amplification techniques in order to detect modified nucleic acid molecules that are produced as a direct result of the action of nucleic acid modifying activity on substrate nucleic acid molecules, which indicates the presence of a microorganism in the sample. In certain embodiments, the technique used is selected from PCR, NASBA, 3SR, TMA, SDA, and DNA oligomer self-assembly.

Detection of the amplification product may be by conventional methods, such as gel electrophoresis, but in some embodiments is performed using real-time or end-point detection methods.

Various techniques for real-time or end-point detection of the products of an amplification reaction are known in the art. These include the use of intercalating fluorescent dyes, such as SYBR Green I (Sambrook and Russell, molecular cloning-A laboratory Manual, third edition), which allow the yield of amplified DNA to be estimated from the amount of fluorescence generated. Many real-time detection methods produce fluorescent readings, which can be continuously monitored. Specific examples include molecular beacons and fluorescence resonance energy transfer probes. Real-time and end-point techniques are advantageous because they keep the reaction in a "single tube". This means that results can be obtained without downstream analysis, and thus results can be obtained more quickly. Furthermore, keeping the reaction in a "single tube" environment reduces the risk of cross-contamination and allows for quantitative output of the method of the invention. This is particularly important in the context of the present invention where health and safety concerns may be of critical importance (e.g. in detecting a potential microbial infection in a patient sample).

Can use

The system (Applied Biosystems) accomplishes real-time and end-point quantification of PCR reactions, see Holland et al, by detecting specific polymerase chain reaction products using the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase, Proc. Natl. Acad. Sci. USA88, 7276-; gelmini et al, homogeneous assay based on quantitative polymerase chain reaction using fluorescent probes to measure C-Erb-2 oncogene amplification, Clin. chem.43,752-758(1997), and Livak et al, go to fully automated genome wide polymorphism screening, nat. Genet.9, 341-342(19995) (incorporated herein by reference). This type of probe may be generally referred to as a hydrolysis probe. Suitable hydrolysis/Taqman probes for real-time or end-point detection are also provided. The probe may be suitably labelled, for example using a label as described in detail below.

In molecular beacon systems, see Tyagi and Kramer, molecular beacon-probes that fluoresce upon hybridization, nat biotechnol.14, 303-308(1996) and Tyagi et al, multicolor molecular beacons for distinguishing alleles, nat biotechnol.16, 49-53(1998) (incorporated herein by reference), beacons are hairpin-shaped probes with internally quenched fluorophores whose fluorescence is restored when bound to their target. These probes may be referred to as hairpin probes.

Another real-time fluorescence based system that can be incorporated into the methods of the present invention is the Scorpion system, see detection of PCR products using self-probing amplicons and fluorescence, Whitcomb et al, Nature Biotechnology 17, 804-. Other real-time or end-point detection techniques well known to those skilled in the art and commercially available include

The technology,

Primer technology, DzyNA primers (Todd et al, J.Clin.Chem.46: 5, 625-.

Thus, in other aspects of the invention, real-time or end-point techniques are used to detect the products of nucleic acid amplification. In particular embodiments of the invention, the real-time technique includes the use of any of the following: hydrolysis probe (

System), FRET probe: (

System), hairpin primer: (

System), hairpin probe (molecular beacon system), hairpin probe incorporated into primer: (

Probe systems), primers incorporating complementary sequences of DNAzyme and cleavable fluorescent DNAzyme substrate (DzYNA), Plexor qPCR, and oligonucleotide blocking systems.

The amplification products can be quantified to give an approximation of the nucleic acid modifying activity of the microorganism in the sample and thus the level of the microorganism in the sample. Thus, "presence or absence" is intended to encompass quantification of the level of a microorganism in a sample.

The inventors further found that the optimal temperature for measuring the nucleic acid modifying activity of a microorganism may be different from the optimal temperature for lysis of the microorganism. Thus, in some embodiments, microbial lysis is performed at a lower temperature than the step of incubating the lysate with a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of the microorganism. As already discussed, in some embodiments of the invention, the substrate nucleic acid molecule is comprised in a lysis reagent for lysing a microorganism. Such embodiments are consistent with different temperature preferences. Thus, even though the substrate nucleic acid molecule may be comprised in the lysis reagent, the initial lower temperature does not adversely affect the subsequent incubation at the higher temperature at which the substrate is modified by the nucleic acid modifying activity released from the microorganism. Thus, in some embodiments, the method comprises a step of lysing the microorganism, wherein the lysis reagent comprises a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of said microorganism. This step is performed at a lower temperature than the subsequent step of incubating the lysate with the substrate nucleic acid molecules, so as to render active the enzymes released from the microorganisms. Thus, the substrate is exposed to an initially lower temperature and then to a higher temperature at which the enzymatic activity is enhanced.

In some embodiments, the step of incubating the lysate with a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of the microorganism is performed at a temperature of at least about 30 ℃. The temperature may optionally be between about 30 ℃ and 40 ℃ or between about 32 ℃ and 37 ℃, e.g., about 37 ℃.

In a further or alternative embodiment, the lysis step of the microorganisms is carried out at a temperature of no more than about 30 ℃, optionally between about 15 ℃ and 30 ℃ or between about 18 ℃ and 25 ℃, for example about 18, 19, 20, 21, 22, 23, 24 or 25 ℃. In some embodiments, all steps prior to incubating the lysate with a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of the microorganism are performed at a temperature of no more than about 30 ℃. The temperature may optionally be between about 15 ℃ to 30 ℃ or between about 18 ℃ to 25 ℃, for example about 18, 19, 20, 21, 22, 23, 24 or 25 ℃.

Such methods may incorporate any one or more up to all of the embodiments described in relation to the various aspects of the invention.

In some embodiments, the method is further characterized in that the step of incubating the lysate with the substrate nucleic acid molecule is performed at a temperature of at least about 30 ℃, optionally between about 30 ℃ and 40 ℃ or between about 32 ℃ and 37 ℃, for example about 37 ℃.

In a further or alternative embodiment, each step prior to incubating the lysate with the substrate nucleic acid molecule is performed at a temperature of no more than about 30 ℃, optionally between about 15 ℃ and 30 ℃ or between about 18 ℃ and 25 ℃, e.g., 18, 19, 20, 21, 22, 23, 24, or 25 ℃.

Prior to the step of incubating the sample with magnetic particles to form particle-microorganism complexes (i.e. prior to step (a)), the method may comprise selectively lysing non-microorganism cells in the sample while leaving microorganisms present in the sample intact.

The step of selectively lysing non-microbial cells in the sample while leaving intact microorganisms present in the sample may comprise adding to the sample a combination of a detergent and one or more enzymes. The one or more enzymes may comprise a protease and/or a dnase, optionally wherein the protease is proteinase K.

The step of selectively lysing non-microbial cells in the sample while leaving intact any microorganisms present in the sample may prevent enzymatic activity from the non-microbial cells (e.g., leukocytes), falsely indicating the presence of microorganisms in the sample. Such selective cleavage can be achieved by any suitable means as further discussed herein. Any reagent that can lyse non-microorganisms, particularly mammalian cells, present in a sample but does not lyse the microorganisms in the sample can be used. In some embodiments, the reagent may include a surfactant or detergent, such as a non-ionic detergent. Suitable examples include, for example, 5% w/v polyethylene glycol sorbitan monolaurate (Tween 20). The agent may comprise, for example, 5% w/v saponin. The reagent may comprise, for example, 8.5g/l of a metal halide salt, such as sodium chloride. The reagent may comprise a mixture of all three components. The sample may be mixed with the reagent under suitable conditions to ensure that non-microbial cells, in particular mammalian cells (if present in the sample), are lysed, but that the microorganisms (if present in the sample) are not (significantly) lysed. The sample may be exposed to the reagent for a period of about 5 to 30min, for example 5, 10, 15, 20, 25 or 30 min. This step may be performed at any suitable temperature, for example between 15 and 30 degrees celsius or at room temperature.

In some embodiments, according to all aspects of the invention, the selective lysis of non-microbial cells in the sample while maintaining any intact microorganisms present in the sample comprises adding to the sample a combination of a detergent and one or more enzymes. Without wishing to be bound by any particular theory, the detergent selectively permeates the non-microbial cell membrane, while the microorganism is protected by its cell wall. The enzyme may be used to break down released intracellular material and other cellular debris and may help prevent the continued presence of released enzyme activity. In some embodiments, the one or more enzymes comprise a protease and/or a nuclease. Suitable proteases include proteinase K. Suitable nucleases include dnase. In one embodiment, the reagent for selectively lysing non-microbial cells comprises a combination of triton X-100 and proteinase K. More specifically, the lysis reagent may comprise 0.25% Triton X-100 and 4.8. mu.g/mL proteinase K.

If the non-microbial cells are lysed, it is important to inactivate any relevant enzyme activity that is released. The inventors have devised a method that utilizes high pH conditions to ensure effective inactivation of enzyme activity. The microbial cells are generally left intact, at least during some treatments, and the high pH treatment does not significantly adversely affect intracellular enzyme activity. In addition, the inventors have previously shown that in any case, microbial enzymes are more resistant to high pH treatment.

Thus, after the step of selectively lysing non-microbial cells in the sample while leaving intact any microorganisms present in the sample, the method may comprise exposing the lysate to high pH conditions. The duration of exposure to the high pH condition is typically less than 20 minutes, and may not exceed 10, 9, 8, 7, 6, or 5 minutes, and may be about 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, treatment is performed for about 2 to 15min, for example about 5 min. "about" means plus or minus 30 seconds.

To provide high pH conditions, any suitable reagent may be used. In particular embodiments, the high pH conditions comprise contacting the sample with a base or buffer. In a specific embodiment, NaOH or Na is used₂CO₃. In particular embodiments, NaOH or Na₂CO₃Is about 5mM or higher. BufferThe liquid may have a pKa value greater than 9. Examples of suitable buffers include borate, carbonate and pyrophosphate buffers.

High pH conditions typically inhibit the activity of nucleic acid modifying enzymes including ATP-dependent ligases and polymerases from non-microbial sources, such as mammalian cells, but do not inhibit the activity of microbial ligases or polymerases. This is mainly due to the differential cleavage conditions employed in the process to ensure that only non-microbial enzymes are exposed to high pH conditions. However, this may also be due to the greater resistance of microbial enzymes to these conditions. The "high pH" is typically a pH of at least about 10, for example about 10, 11, 12, 13 or 14. "Low pH" is typically a pH of less than or equal to about 4, for example about 4, 3, 2 or 1. By "about" is meant 0.5 of the pH unit on either side of the stated value. As will be readily appreciated by those skilled in the art, any suitable means may be used to effect a change in the pH of the sample. Microbial enzymes such as polymerases and ligases may be resistant to extreme pH, while corresponding mammalian enzymes may be inactivated under the same pH conditions. This facilitates the selective detection of microbial enzyme activity in a sample comprising mammalian cells and microbial cells. In certain embodiments, the conditions that inhibit non-microbial nucleic acid modifying activity, e.g., the activity of an ATP-dependent ligase, but do not inhibit nucleic acid modifying activity, e.g., microbial-derived activity of a microbial ligase, from mammalian cells comprise use of sodium hydroxide (NaOH) or sodium carbonate (Na)₂CO₃) The sample is processed. As shown herein, such reagents can be readily used to increase the pH of a sample to a high pH, thereby inactivating non-microbial enzyme activity while maintaining microbial (fungal and bacterial) enzyme activity. Suitable concentrations and volumes of suitable reagents may be applied by the skilled person. However, in certain embodiments, the NaOH is at least about 5mM NaOH. In some embodiments, the base concentration is no more than 10mM, e.g., 5, 6, 7, 8, 9, or 10 mM.

In other embodiments, the pH is about 12 to inactivate mammalian nucleic acid modifying activity (e.g., polymerase and/or ATP-dependent ligase activity) but not microbial nucleic acid modifying activity (e.g., polymerase and/or ligase activity). In particular embodiments, the pH conditions may be increased to at least about 11 or at least 11.2. After a certain period of time, this treatment may result in the lysis of the microorganisms in the sample and thus in the release of nucleic acid modifying activity (e.g. polymerase and/or ligase) into the sample. Thus, in some embodiments, lysis of the microorganism is achieved by high pH treatment. This allows detection of nucleic acid modifying activity (e.g. polymerase and/or ligase) derived from the microorganism in the sample without the need for a separate cell lysis step. Under these conditions, mammalian ligases (e.g., blood ATP-dependent ligases) are inactivated. However, as discussed in more detail herein, typically, the method comprises a separate step for lysing the microorganisms in the sample.

In some embodiments, treatment under high pH conditions is stopped by adding a pH-lowering agent. This is done prior to lysis of the microorganisms. Suitable reagents include buffers and/or acids. Thus, the pH can be lowered by adding a neutralization buffer. In particular embodiments, the buffer comprises a Tris-HCl buffer (e.g., pH 7.2 or 8). Other suitable agents for lowering the pH include acids such as hydrochloric acid (HCl) and sulfuric acid (H)₂SO₄). These (and other) acids may be incorporated into the buffer as will be readily understood by those skilled in the art. One particular reagent that may be used to process the sample after the pH is raised includes a combination of ammonium sulfate, magnesium sulfate heptahydrate, potassium chloride, and Tris-HCl. More specifically, the reagent may contain 10mM ammonium sulfate, 2mM magnesium sulfate heptahydrate, 10mM potassium chloride and 20mM Tris-HCl [ pH8.0]]。

Step (b) may further comprise removing non-microbial cells from the particle-microbe complex, for example by aspiration.

In the context of the present invention, a "sample" is a sample that contains non-microbial cells and is intended to be tested for the presence of a microorganism, such as a fungus (e.g., yeast) and/or a bacterium, that expresses a nucleic acid modifying activity. Thus, a sample may comprise, consist essentially of or consist of: clinical samples, such as blood samples (including whole blood, plasma, serum and blood-containing samples, such as blood cultures or blood culture solutions). The method of the invention is particularly suitable for the rapid determination of negative (and positive) blood cultures. Thus, the sample may comprise a blood culture sample (or blood broth sample) from a patient suspected of having or being screened for a blood stream infection. The sample may be any suitable volume, for example 1 to 10ml, preferably 1ml of a blood culture sample.

The sample used will depend on a variety of factors such as availability, convenience and conditions to be tested. Typical samples that may be used, but are not intended to limit the present invention, include whole blood, serum, plasma, platelets, synovial fluid, urine samples, and the like, taken from a patient, most preferably a human patient. The patient may be suspected of having a blood infection or is undergoing a blood infection screen. The patient may be a hospitalized patient. Samples may be taken from subjects containing more than 5, 10, or 1500 million White Blood Cells (WBCs) per milliliter of blood. The methods of the invention represent in vitro tests. They are performed on samples taken from subjects. However, in a less preferred embodiment, the method may additionally comprise the step of obtaining a sample from the subject. Methods for obtaining a suitable sample from a subject are well known in the art. In general, however, the method may be performed starting from a sample that has been isolated from the patient in a separate procedure. The method will most preferably be performed on a sample from a human, but the method of the invention can be used on many animals.

The method of the invention may be used to supplement any diagnostic technique already available, possibly as a method of confirming an initial diagnosis. Alternatively, the method itself may be used as a preliminary diagnostic method, as it provides a rapid and convenient diagnostic means. Furthermore, due to its inherent sensitivity, the method of the present invention requires only a minimal sample, thereby preventing unnecessary invasive surgery. In addition, large but unconcentrated samples can also be effectively tested according to the methods of the present invention.

In a particular embodiment according to all aspects of the invention, the microorganism that can be detected in the sample is a pathogenic microorganism, such as a pathogenic bacterium or a fungus/yeast. The bacteria may be any bacteria capable of causing an infection or disease in a subject, preferably a human subject. In one embodiment, the bacteria comprise, consist essentially of, or consist of one or more of the following: staphylococcal (Staphylococcus) species, including Staphylococcus epidermidis (Staphylococcus epidermidis) and Staphylococcus aureus (Staphylococcus aureus) (and preferably methicillin-resistant strains), enterococcal (Enterococcus) species, Streptococcus (Streptococcus) species, Mycobacterium (Mycobacterium) species, particularly Mycobacterium tuberculosis (Mycobacterium tuberculosis), Vibrio (Vibrio) species, particularly Vibrio cholerae (Vibrio cholerae), Salmonella (Salmonella) and/or Escherichia coli (Escherichia coli), and the like. In certain embodiments, the bacteria may comprise, consist essentially of, or consist of: clostridium (Clostridium) species, and in particular Clostridium difficile (c.difficile). Difficile is a major cause of antibiotic-associated diarrhea and colitis, which are medically-associated intestinal infections, primarily affecting elderly patients with other potential diseases. Candida species, such as Candida albicans (c.albicans), Candida parapsilosis (c.parapsilosis) and Candida glabrata (c.glabrata), can be detected. Cryptococcus species, such as Cryptococcus neoformans (c. neoformans), can be detected. Mycosis, such as candidemia, can be detected (or not) using the present invention. Preferably (although not necessarily) the microorganism is indicated by its enzymatic activity. Thus, these methods provide an indication of viable or recent microorganisms in the sample. After a period of time, if the microorganism is not viable, the enzyme activity will disappear from the sample. This represents an advantage of using enzymatic activity over using nucleic acid molecules, in particular DNA, which can last longer, as an indicator of the microorganisms in the sample.

Once a positive presence of a microorganism is detected in a sample, the methods of the invention may involve identifying the nature of the infection. Any suitable method may be used for this further identification step.

The magnetic particles may be superparamagnetic particles.

The particles (e.g. magnetic particles) may have a greater affinity for microorganisms than for non-microbial cells. The magnetic particles may bind to the microorganisms by non-specific binding.

The particles (e.g., magnetic particles) can have an outer polymer surface comprising polystyrene and/or poly (styrene/divinylbenzene).

The magnetic particles may comprise iron oxide. Preferably, the iron oxide is encapsulated by the outer polymer surface. The particles may be an amalgam of iron oxide and polymer. The particles may be partially encapsulated by the outer polymer surface. The polymer may comprise polystyrene.

The particles (e.g. magnetic particles) may have a diameter between 0.05 and 1 μm, between 0.1 and 0.5 μm, between 0.2 and 0.3 μm. Preferably, the magnetic particles may have a diameter between 0.2 and 0.3 μm. The particles (e.g. magnetic particles) may have a diameter between 0.1 and 3 μm or 0.1 and 2 μm. More preferably, the particles have a diameter between 0.1 and 1.0 μm.

The particles (e.g. magnetic particles) may have an outer polymer surface. The outer polymer surface of the magnetic particles must not be coated with any of the following: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin protein, (v) a polyamine or (vi) a cationic detergent.

The Mannose Binding Lectin (MBL) protein may be an MBL-based genetically engineered protein. For example, it may be a genetically engineered protein comprising a pathogen-binding portion of MBL (i.e., FcMBL) fused to the Fc region of an immunoglobulin.

The outer surface (e.g., outer polymer surface) of the particle (e.g., magnetic particle) may not be coated with any of the following: (i) an antibody, (ii) a carbohydrate, or (iii) an innate immune system protein.

The outer surface (e.g., outer polymer surface) of the particle (e.g., magnetic particle) may not be coated with any of the following: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin, or (v) a flocculant agent (e.g. as defined in WO 03/102184).

The outer surface (e.g., outer polymer surface) of the particle (e.g., magnetic particle) may not be coated with any of the following: (i) an antibody, (ii) a carbohydrate, (iii) an innate immune system protein or (iv) a flocculant (e.g., a flocculant as defined in WO 03/102184).

The antibody may be a fragment or derivative of an antibody that retains the antigen-specific binding function. Such fragments and derivatives include Fab fragments, ScFv, single domain antibodies, nanobodies, heavy chain antibodies, and the like.

The carbohydrate may be a monosaccharide, oligosaccharide (e.g. disaccharide or trisaccharide), polysaccharide and/or a derivative thereof.

The outer surface (e.g., outer polymer surface) of the particle (e.g., magnetic particle) may not be coated with the ligand. The outer surface (e.g. outer polymer surface) of the particles (e.g. magnetic particles) may not be coated with non-specific ligands (e.g. non-specific ligands as described in WO 01/53525). The outer surface (e.g. outer polymer surface) of the particles (e.g. magnetic particles) may not be coated with non-protein ligands (e.g. non-protein ligands as described in WO 01/53525).

The outer surface (e.g., outer polymer surface) of the particles (e.g., magnetic particles) may be carboxylated. The outer surface (e.g. outer polymer surface) of the particles (e.g. magnetic particles) may be coated with only carboxyl groups.

The outer surface (e.g., outer polymer surface) of the particles (e.g., magnetic particles) can be coated with streptavidin. The outer surface (e.g., outer polymer surface) of the particle (e.g., magnetic particle) can be coated with streptavidin and not with ligands. The outer surface (e.g., outer polymer surface) of the particle (e.g., magnetic particle) may be coated with only streptavidin.

The outer surface (e.g., outer polymer surface) of the particles (e.g., magnetic particles) may not be coated.

The microorganism can be a pathogenic microorganism, optionally wherein the pathogenic microorganism is a pathogenic bacterium or fungus.

The non-microbial cells may include red blood cells and/or white blood cells.

The invention further provides a composition. The compositions provided herein can be used to perform any of the methods described herein. All aspects and embodiments described with respect to the method of the invention apply mutatis mutandis to the relevant composition.

The composition may comprise: i) a particle capable of forming a complex with a microorganism, wherein the particle has an outer surface; ii) sodium polyanetholesulfonate; and iii) at least one agent that selectively lyses non-microbial cells in the sample while leaving intact microorganisms present in the sample.

The composition may comprise: i) a particle capable of forming a complex with a microorganism, wherein the particle has an outer surface; ii) sodium polyanetholesulfonate; and iii) a detergent. Detergents are examples of reagents that selectively lyse non-microbial cells in a sample while leaving intact the microorganisms present in the sample.

The composition may further comprise microbial cells and/or non-microbial cells. The composition may comprise a sample suspected of containing microbial cells and known to contain non-microbial cells.

The composition may further comprise a buffer and/or sodium chloride.

In this composition, the reagent that selectively lyses non-microbial cells in the sample while leaving intact the microorganisms present in the sample can be a detergent. Optionally wherein the detergent is non-ionic. In the composition, the detergent may not be conjugated to a particle capable of forming a complex with the microorganism. Thus, typically the detergent forms part of the solution to which the particles are added, and not of the particles themselves.

In the composition, the particles may have a diameter of between 0.1 and 3 μm, or between 0.1 and 2 μm. Preferably, the particles have a diameter between 0.1 and 1.0 μm.

In this composition, the particles may be (and typically are) magnetic. In this method, the particles may be superparamagnetic. The particles may comprise iron oxide. The iron oxide may include magnetite and/or maghemite. The iron oxide may not contain Fe in a 1:1, 2:1, 3:1, or 4:1 ratio²⁺And Fe³⁺。

In the composition, the outer surface of the particle capable of forming a complex with the microorganism may comprise a polymer; optionally, the polymer may be carbon-based. The polymer may not comprise an inorganic polymer. The polymer may comprise polystyrene and/or poly (styrene/divinylbenzene).

In the composition, the outer surface of the particles capable of forming a complex with the microorganism may comprise or be coated with any one or more of: i) a carboxylic acid group; ii) an amino group; iii) a hydrophobic group; and iv) streptavidin; optionally, a carboxylic acid group; ii) an amino group; iii) the hydrophobic group may not be part of the polypeptide.

In the composition, the microorganism may be a pathogenic microorganism. For example, the pathogenic microorganism may be a pathogenic bacterium or fungus.

In the composition, the non-microbial cells can include red blood cells and/or white blood cells.

In this composition, the sample may comprise blood, urine, saliva or milk, optionally wherein the sample is whole blood.

The invention further provides kits for performing any of the methods described herein. All aspects and embodiments described in relation to the method of the invention apply mutatis mutandis to the relevant kit.

The kit may comprise: i) a particle capable of forming a complex with a microorganism, wherein the particle has an outer surface; ii) sodium polyanetholesulfonate; and iii) at least one agent that selectively lyses non-microbial cells in the sample while leaving intact microorganisms present in the sample.

The kit may comprise: i) a particle capable of forming a complex with a microorganism, wherein the particle has an outer surface; ii) sodium polyanetholesulfonate; and iii) a detergent. The detergent is an example of selectively lysing non-microbial cells in a sample while leaving intact the microorganisms present in the sample.

The detergent may be non-ionic. The detergent may not be conjugated to a particle capable of forming a complex with the microorganism. Thus, typically the detergent forms part of the solution to which the particles are added, and not of the particles themselves.

In the kit, the particles may have a diameter between 0.1 and 3 μm or between 0.1 and 2 μm. Preferably, the particles have a diameter between 0.1 and 1.0 μm.

In this kit, the particles may be (and typically are) magnetic. The particles may be superparamagnetic. The particles may comprise iron oxide. The iron oxide may include magnetite and/or maghemite. The iron oxide may not contain Fe in a 1:1, 2:1, 3:1, or 4:1 ratio²⁺And Fe³⁺。

The outer surface of the particle may comprise a polymer; preferably, the polymer may comprise a polymer. Optionally, the polymer may be carbon-based. The polymer may not comprise an inorganic polymer. The polymer may comprise polystyrene and/or poly (styrene/divinylbenzene).

The kit may comprise: a) particles capable of forming a complex with a microorganism; b) sodium polyanetholesulfonate; c) at least one agent that selectively lyses non-microbial cells in the sample while leaving intact microorganisms present in the sample; and d) detection means for detecting the presence or absence of a microorganism in the particle-microorganism complex, wherein the detection means comprises a nucleic acid molecule which serves as a substrate for the nucleic acid modifying activity of the microorganism, and wherein the nucleic acid molecule is at least partially double-stranded and comprises a uracil residue in the complementary strand.

The kit may comprise: a) particles capable of forming a complex with a microorganism; b) sodium polyanetholesulfonate; c) a detergent; and d) detection means for detecting the presence or absence of a microorganism in the particle-microorganism complex, wherein the detection means comprises a nucleic acid molecule serving as a substrate for the nucleic acid modifying activity of the microorganism, and wherein the nucleic acid molecule is at least partially double-stranded and comprises a uracil residue in the complementary strand.

The kit may comprise: a) a particle capable of forming a complex with a microorganism, wherein the outer surface of the particle is not coated with any one of: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from apolipoprotein H, (iv) a mannose-binding lectin protein; and b) detection means for detecting the presence or absence of a microorganism in the microparticle-microorganism complex, wherein the detection means comprises a nucleic acid molecule that serves as a substrate for the nucleic acid modification activity of the microorganism, and wherein the nucleic acid molecule is at least partially double-stranded and comprises a uracil residue in the complementary strand.

The kit may further comprise reagents for selectively lysing non-microbial cells in the sample while leaving intact the microorganisms present in the sample.

The kit may further comprise a buffer and/or sodium chloride.

In this kit, the reagent that selectively lyses non-microbial cells in the sample while leaving intact the microorganisms present in the sample can be a detergent. Optionally, the detergent may be non-ionic. In the composition, the detergent may not be conjugated to a particle capable of forming a complex with the microorganism.

In the kit, the particles may have a diameter between 0.1 and 3 μm, between 0.1 and 2 μm. Preferably, the particles have a diameter between 0.1 and 1.0 μm.

In the kit, the particles may be magnetic. In this method, the particles may be superparamagnetic.

In the kit, the outer surface of the particle capable of forming a complex with the microorganism may comprise a polymer; optionally, the polymer may be carbon-based.

In this kit, the outer surface of the particle capable of forming a complex with a microorganism may comprise or be coated with any one or more of: i) a carboxylic acid group; ii) an amino group; iii) a hydrophobic group; and iv) streptavidin; optionally a carboxylic acid group; ii) an amino group; iii) the hydrophobic group may not be part of the polypeptide.

In this kit, the microorganism can be a pathogenic microorganism, optionally wherein said pathogenic microorganism can be a pathogenic bacterium or fungus.

In this kit, the non-microbial cells may comprise red blood cells and/or white blood cells.

In this kit, the sample may comprise blood, urine, saliva or milk, optionally wherein the sample is whole blood.

The kit may comprise (a) particles (e.g. magnetic particles) capable of forming a complex with the microorganism; and (b) detection means for detecting the presence or absence of microorganisms in said microparticle-microorganism complex.

Any suitable detection means may be employed and they may represent the complete set of reagents required to detect the presence or absence of microorganisms in the particle-microorganism complex.

In certain embodiments, the detection means comprises or is a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of said microorganism.

In some embodiments, the detection means comprises or further comprises reagents for nucleic acid amplification. The reagents for nucleic acid amplification may comprise a primer pair and/or at least one probe. In some embodiments, those primers and/or probes hybridize to microbial nucleic acid molecules. Thus, they can detect microorganisms in a sample by detecting amplified microbial nucleic acid molecules. Alternatively, the primer or probe hybridizes to a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of the microorganism. Such nucleic acid molecules are described in further detail herein.

The kit may comprise: (a) particles (e.g. magnetic particles) capable of (selectively) forming complexes (e.g. particle-microorganism complexes) with microorganisms; and (b) detection means for detecting the presence or absence of microorganisms in the particle-microorganism complex. The detection means may comprise a nucleic acid molecule that acts as a substrate for the nucleic acid modifying activity of the microorganism. The nucleic acid molecule may be at least partially double stranded, and may optionally comprise a uracil residue in the complementary strand. The complementary strand may comprise a base (e.g., dideoxycytidine) at its 3' end that blocks DNA polymerase-mediated elongation of the second strand. The nucleic acid molecule can be any nucleic acid molecule described herein.

In this kit, the outer surface (e.g., outer polymer surface) of the particles (e.g., magnetic particles) may not be coated with any of the following: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from apolipoprotein H, (iv) a mannose-binding lectin protein, (v) a polyamine or (vi) a cationic detergent.

In this kit, the outer surface (e.g., outer polymer surface) of the particles (e.g., magnetic particles) may not be coated with any of the following: (i) an antibody, (ii) a carbohydrate, or (iii) an innate immune system protein.

In this kit, the outer surface (e.g., outer polymer surface) of the particles (e.g., magnetic particles) may not be coated with any of the following: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from apolipoprotein H, (iv) a mannose-binding lectin, or (v) a flocculant agent (e.g. as defined in WO 03/102184).

In this kit, the outer surface (e.g., outer polymer surface) of the particles (e.g., magnetic particles) may not be coated with any of the following: (i) an antibody, (ii) a carbohydrate, (iii) an innate immune system protein or (iv) a flocculant (e.g. as defined in WO 03/102184).

In this kit, the particles (e.g., magnetic particles) can have an outer surface (e.g., outer polymer surface) that is not coated with ligands.

In this kit, the particles (e.g., magnetic particles) can have an outer surface (e.g., outer polymer surface) that is coated with streptavidin and not with ligands.

In this kit, the particles (e.g., magnetic particles) can have an outer surface (e.g., outer polymer surface) coated with only streptavidin.

In this kit, the particles (e.g., magnetic particles) may have an outer surface (e.g., outer polymer surface) that is coated with only carboxyl groups.

In this kit, the particles (e.g., magnetic particles) can have an outer surface (e.g., an outer polymer surface) that is not coated with any molecules or moieties capable of binding microorganisms.

The (substrate) nucleic acid molecule may be designed based on the inclusion of a DNA or RNA polymerase enzyme by the nucleic acid modifying enzyme. In some embodiments, the DNA polymerase is DNA polymerase I. In other or alternative embodiments, the nucleic acid modifying enzyme comprises a ligase, such as an ATP or NAD-dependent ligase.

The detection means may further comprise reagents for nucleic acid amplification, optionally wherein the reagents for nucleic acid amplification comprise a primer pair and/or at least one probe that hybridizes to the nucleic acid molecule.

The kit may further comprise a reagent capable of lysing the microorganisms in the particle-microorganism complex, optionally wherein the reagent capable of lysing the microorganisms in the particle-microorganism complex comprises a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of said microorganisms.

The reagent that selectively lyses non-microbial cells in a sample while leaving intact microorganisms present in the sample may comprise a combination of a detergent and one or more enzymes, wherein the one or more enzymes optionally comprise a protease and/or a dnase. Suitable detergents and enzymes are discussed herein.

The kit may further comprise a high pH reagent, such as a base or buffer. This may be, for example, NaOH, e.g. 5mM NaOH. Other suitable agents are described herein.

The kit may further comprise a neutralization buffer. The neutralization buffer may be capable of restoring the pH of the sample after the high pH treatment. Suitable agents are described herein.

The nucleic acid modifying enzyme may comprise: (a) a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I; and/or (b) a ligase, optionally wherein the ligase is an ATP and/or NAD dependent ligase.

According to all relevant aspects and embodiments of the present invention, the term "polyanetholesulfonic acid sodium" is intended to cover all functionally equivalent derivatives and salt forms thereof (e.g. polyanetholesulfonic acid potassium, polyanetholesulfonic acid magnesium, etc.).

Throughout the disclosure, the terms "particle" and "bead" may be used interchangeably.

The invention may also be defined by the following clauses:

1. a method of separating microorganisms from non-microbial cells in a sample containing the non-microbial cells, the method comprising:

a) incubating the sample with magnetic particles to form particle-microorganism complexes; and

b) separating the particle-microorganism complexes from the non-microbial cells using a magnetic field, wherein the magnetic particles have an outer polymer surface that is not coated with any one of: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin protein, (v) a polyamine or (vi) a cationic detergent.

2. A method of detecting the presence or absence of a microorganism in a sample that may also contain non-microbial cells, comprising:

a) incubating the sample with magnetic particles to form particle-microorganism complexes;

b) separating the particle-microorganism complex from the non-microbial cell using a magnetic field; and

c) detecting the presence or absence of microorganisms in the particle-microorganism complex

Wherein the magnetic particles have an outer polymer surface that is not coated with any one of: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin protein, (v) a polyamine or (vi) a cationic detergent.

3. The method of clause 2, wherein step (c) comprises (i) detecting the enzymatic activity of a nucleic acid molecule associated with the microorganism, (ii) detecting the microorganism directly by cell counting or microscopy, or (iii) detecting the microorganism after cell culture.

4. The method of clause 2 or clause 3, wherein step (c) comprises the steps of:

i. lysing the microorganisms in the particle-microorganism complex;

incubating the lysate with a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of the microorganism; and

specifically determining the presence or absence of a modified nucleic acid molecule produced by the action of the nucleic acid modifying enzyme on said substrate nucleic acid molecule to indicate the presence or absence of a microorganism.

5. The method of clause 4, wherein step (i) comprises adding a lysis reagent comprising a substrate nucleic acid molecule.

6. The method according to clause 4 or clause 5, wherein the nucleic acid modifying enzyme comprises a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I.

7. The method according to any of clauses 4 to 6, wherein the nucleic acid modifying enzyme comprises a ligase, optionally wherein the nucleic acid modifying enzyme is an NAD-dependent ligase.

8. A method of detecting the presence or absence of a microbial infection in a subject, comprising performing the method of any one of clauses 2 to 7 on a sample from the subject.

9. The method of any one of the preceding clauses, wherein the method further comprises washing the isolated particle-microorganism complex to remove non-microbial cells or lysate.

10. The method of any one of the preceding clauses wherein prior to step (a), the method comprises selectively lysing non-microbial cells in the sample while leaving intact microorganisms present in the sample.

11. The method of clause 10, wherein selectively lysing non-microbial cells in the sample while leaving intact any microbes present in the sample comprises adding to the sample a combination of a detergent and one or more enzymes; wherein the one or more enzymes comprise a protease and/or a dnase, optionally wherein the protease is proteinase K.

12. The method of any one of the preceding clauses wherein step (b) further comprises removing the non-microbial cells from the particle-microbe complex.

13. The method of any one of the preceding clauses wherein the sample comprises blood.

14. A kit for performing the method of any of clauses 4 to 13, comprising:

a) magnetic particles capable of forming a complex with a microorganism, wherein the outer polymer surface of the magnetic particles is not coated with any one of: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin protein, (v) a polyamine or (vi) a cationic detergent; and

b) detection means for detecting the presence or absence of a microorganism in a particle-microorganism complex, wherein the detection means comprises a nucleic acid molecule which serves as a substrate for the nucleic acid modifying activity of said microorganism, and wherein said nucleic acid molecule is at least partially double-stranded and comprises a uracil residue in the complementary strand.

15. The kit of clause 14, wherein the detection means further comprises reagents for nucleic acid amplification, optionally wherein the reagents for nucleic acid amplification comprise a primer pair and/or at least one probe that hybridizes to the nucleic acid molecule.

16. The kit of clause 14 or clause 15, further comprising a reagent capable of lysing the microorganisms in the particle-microorganism complex, optionally wherein the reagent capable of lysing the microorganisms in the particle-microorganism complex comprises a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of the microorganisms.

17. The kit of any of clauses 14 to 16, further comprising reagents that selectively lyse non-microbial cells in the sample while leaving intact microorganisms present in the sample.

18. The kit of clause 17, wherein the reagent that selectively lyses non-microbial cells in the sample while leaving intact microorganisms present in the sample comprises a combination of a detergent and one or more enzymes, wherein the one or more enzymes optionally comprise a protease and/or a dnase.

19. The kit of any one of clauses 14 to 18, further comprising:

a) a high pH agent; and/or

b) And (4) neutralizing the buffer solution.

20. The kit of any one of clauses 14 to 19, wherein the sample comprises blood.

21. The kit of any one of clauses 14 to 20, wherein the nucleic acid modifying enzyme comprises:

a) a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I; and/or

b) A ligase, optionally wherein the ligase is an ATP and/or NAD dependent ligase.

22. The method of any one of clauses 1 to 13, or the kit of any one of clauses 14 to 21, wherein the magnetic particles are superparamagnetic particles.

23. The method of any one of clauses 1 to 13 or 22, or the kit of any one of clauses 14 to 22, wherein the outer polymer surface comprises polystyrene.

24. The method of any one of clauses 1 to 13 or clauses 22 or 23, or the kit of any one of clauses 14 to 23, wherein the magnetic particles comprise iron oxide.

25. The method of any one of clauses 1 to 13 or clauses 22 to 24, or the kit of any one of clauses 14 to 24, wherein the magnetic particles have a diameter of between 0.1 to 0.5 μ ι η.

26. The method of any one of clauses 1 to 13 or clauses 22 to 25, or the kit of any one of clauses 14 to 25, wherein the outer polymer surface of the magnetic particles is coated with streptavidin.

27. The method of any one of clauses 1 to 13 or clauses 22 to 26, or the kit of any one of clauses 14 to 26, wherein the outer polymer surface of the magnetic particles is carboxylated.

28. The method or kit of clause 26, wherein the outer polymer surface of the magnetic particle is coated with streptavidin and not with a ligand.

29. The method of any one of clauses 1 to 13 or clauses 22 to 27, or the kit of any one of clauses 14 to 27, wherein the outer polymer surface of the magnetic particle is not coated with a ligand.

30. The method of any one of clauses 1 to 13 or 22 to 29, or the kit of any one of clauses 14 to 29, wherein the microorganism is a pathogenic microorganism, optionally wherein the pathogenic microorganism is a pathogenic bacterium or fungus.

31. The method of any one of clauses 1 to 13 or clauses 22 to 30, or the kit of any one of clauses 14 to 30, wherein the non-microbial cells comprise red blood cells and/or white blood cells.

Drawings

Fig. 1 is an image of a blood sample and shows the degree of blood lysis for each sample group: E-BUF, Urea, Tris + NaCl, frozen (left to right) (see example 8).

Figure 2 is an image of the final sample output before PCR setup. This image provides a visual demonstration of the benefits of SPS on sample processing with magnetic beads in the blood: SPS appears to be able to remove blood components more thoroughly, as indicated by less red eluate in the presence of SPS. Note that the BacTec PLUS aerobic broth used for the blood broth sample set also contained SPS (see example 9).

The invention will be understood with reference to the following non-limiting examples:

experimental part

Abbreviations and definitions:

5 th% fifth percentage threshold calculation to determine 5% FPR (formula: PERCENTILE. INC. (array, 0.05))

BO-only culture solution

BB blood culture solution

Cfu colony Forming Unit

Confirmation of PCR multiplex detection of microbial DNA according to gram conditions (gram-negative, gram-positive or Candida)

CPD citric acid glucose phosphate

Ct cycle threshold

CV cutoff (cfu): based on the theoretical detection limit for cfu value and Δ Ct, using the equation: sample cfu/2^ΔCt

Dilution point (10 times series)

E cfu uses the extrapolated cfu value of the dilution point of the highest countable TVC in the dilution series

EC Escherichia coli

ETGA enzyme template production and amplification

IPC internal process control: PCR template was present in LM to demonstrate correct sample treatment and to verify PCR amplification in ETGA negative samples

Growth of LAWN fusion microorganisms

Microbial lysis mixture comprising a mixture of detergent and microbial lyase

MM mixture (Master Mix)

No amplification beyond threshold fluorescence after 50 cycles without Ct

NSC No-Inclusion (Spike) control

O/n overnight

PC polymerase-inclusion complex control

PCR polymerase chain reaction

Positive threshold value calculated by Pt according to NSC/NC result

qPCR quantitative polymerase chain reaction

RT Room temperature (+19 to +20 ℃ C.)

s/n supernatant

SPS sodium polyanetholesulfonate

TNTC too high to count

Total viable count of TVC

WB wash buffer (containing Tris-HCl + sodium chloride + Igepal + sodium deoxycholate + Tergitol, unless otherwise indicated); or whole blood as noted therein.

The difference between the two Ct values for the Δ Ct (usually the NSC Ct-positive sample Ct)

Example 1

In manual format, two bead types (Merck Bio-Estapor (Streptavidin conjugated) 300nm beads (product-BE-M08/03; "Bio-Estapor") and Ademtech Bio-Adembeads Streptavidin Plus 200nm beads (product number 03222; "Bio-Ademtech")) were compared with ApoH Technologies Peps6 beads (reference-MP 20006; "ApoH Peps 6").

In experiment 1A, binding of aliquots of Bio-Estapor beads (25uL) and aliquots of ApoH Pep6 beads (10uL) was compared. The larger the volume of Bio-Estapor, the lower the number of beads per ml in the material provided compared to ApoH material. Three organisms were tested: escherichia coli (gram negative bacteria), staphylococcus epidermidis (gram positive bacteria) and candida albicans (yeast). 0.5mL of the organism suspension was exposed to magnetic beads in 0.5mL of "TTGB" microorganism binding buffer provided in an ApoH Peps6 kit ("Peps 6 Captobac", reference MP 10031-50T).

After allowing the organisms to bind for 30min, the bead samples were separated from the liquid supernatant by applying a magnetic field to concentrate the beads and removing the supernatant with a pipette. Gently washing the beads with trisections of wash buffer (50mM Tris pH8, 1% v/v Igepal CA-630, 150mM NaCl, 0.25% v/v Tergitol 15-S-9), and the retained supernatant and washed beads were analyzed for viable organisms by two methods; colony counts on agar plates and detection of microbial DNA by Enzymatic Template Generation and Amplification (ETGA) assays (e.g., Zweitzig et al, 2012, characterization of novel DNA polymerase activity assays that allow sensitive, quantitative and universal detection of viable bacteria, nucleic acid studies 40, 14, e109, 1-12; and as described in WO2011/130584, WO2013/103744 and WO 2016/005768).

Plate counts in Table 1A indicate that most of the growth was found from beads (33CFU) and supernatant (2CFU) for Bio-Estapor beads and E.coli, and this is similar to the results for ApoH Peps 6. No growth was found for Staphylococcus epidermidis and the microorganism appeared to have not grown in the original broth. For both Bio-Estapor and ApoH Peps6, Candida albicans showed approximately 10% CFU and 90% binding in the supernatant. These results indicate that under the conditions tested, the Bio-Estapor beads appear to bind organisms at a rate comparable to the rate at which commercially available organism-bound beads, Peps 6. Sensitive ETGA testing supported this result, but showed that staphylococcus epidermidis bound to Bio-Estapor better than Peps6, as shown by the lower Cq values.

TABLE 1A

Experiment 1B shows the binding of E.coli under similar conditions as experiment 1A, although the washing step was omitted in 1B. Experiment 1B shows that another bead Bio-Ademtech also binds to the organism, albeit at a lower level (see table 1B). Here, the plate count indicates that about one third of the liveness counts have bound to the beads. The more sensitive ETGA DNA polymerase assay showed that half of the organisms remained on the beads, as the Cq of the beads and supernatant were approximately equal.

TABLE 1B

Example 2

In experiment 2, ApoH Peps6, Bio-Estapor and Estapor beads (product MI-030/40; "Estapor COOH") with carboxylated surfaces were compared. The number of organisms remaining in the supernatant after 30min of E.coli binding to the beads was measured using a fluorescent ATP assay (BacTiter-Glo microbial cell viability assay; Promega Corporation, G8230). Although this is an indirect test, since it does not allow direct detection of the presence of organisms on the beads, it is a comparable test that is useful for ligand-based beads (ApoH Peps6) and non-ligand beads of the invention (Bio-Estapor and Estapor COOH). Binding to 1mL 10 at 30mL from phosphate saline buffer⁴After CFU/mL E.coli, aliquots of the supernatant were assayed for ATP using the BacTiter-Glo assay as a measure of organism content. The results in Table 2 show that the reduction in organism levels in the supernatants of the Peps6 beads, Bio-Estapor and Estapor COOH was 33%, 27% and 24%, respectively, when measured using this technique.

TABLE 2

Example 3

Example 3 shows the results of testing Escherichia Coli (EC), Staphylococcus Aureus (SA) and Candida Albicans (CA) in a dilution series by the method of magnetic separation described in automated example 1. The assay uses Bio-Estapor beads 300nm in diameter as capture media with binding buffer containing 0.25% Tergitol TTGB. Since each of the three organisms was diluted 10-fold, the continuous change in Ct was recorded, and thus a dose response curve was constructed.

TABLE 3

The following example illustrates the universal microbial capture of microorganisms by magnetic beads in the Momentum's magnet test. The Magnitor test includes two microbiological detection readings:

ETGA: detection of microbial polymerase from intact microbial cells

Confirmation: detection of microbial DNA according to gram-State (gram-negative, gram-positive or Candida)

The main discovery is as follows:

magnetic beads capture bacteria and fungi from simple buffers and various complex biological sample types

Microbial capture using a variety of different bead sizes (0.2 to 1.5 μm diameter beads) and surface coatings (e.g., carboxylated, hydrophobic, aminated, etc.)

Certain binding buffer components can improve microbial detection in the Magnitor assay, such as detergent-based blood lysis.

Example 4: detection of microorganisms relies on the capture of magnetic beads

The target is as follows:

the microbial binding performance of escherichia coli, staphylococcus aureus and candida albicans was evaluated in a simple Tris + NaCl buffer (pH buffered with a physiological saline concentrate to prevent microbial osmotic shock that can occur only in water). A "no bead control" sample set was also included in this experiment to demonstrate that detection is dependent on the presence of magnetic beads for microbial capture.

Preparing magnetic beads:

estapor beads (Merck, Cat # M1-30/40) were washed 3X 1mL in 1 XTiTris + NaCl buffer: 40 u L beads heavy suspension in the final volume of 400 u L1X Tris + NaCl buffer (1% solid content)

The scheme is as follows:

the overnight liquid culture (o/n) of the microorganism was set as a standard in BacTec PLUS aerobic medium (3 mL of medium inoculated from an agar plate). The next day (after about 16 hours) 1.88. mu.L of E.coli and S.aureus broth cultures were added to 3mL of broth, and 18.75. mu.L of C.albicans broth cultures were added to 3mL of broth; and was proliferated at 37 ℃ for 2 hours at 500 rpm.

After 2 hours of growth, the microbial preculture (DF10) was diluted in 1 XTTris + NaCl buffer (50mM Tris-HCl [ pH8.0] +150mM NaCl) to create four dilution points for each microorganism.

100 μ L TVC for each microbial dilution

Manual simulation of the magnet was performed using DynaMag-2 magnets and manual liquid delivery:

add 1mL of sample to 2mL tube containing 15 μ L of prewashed beads-note that 112 μ L of 1 × Tris + NaCl buffer was not added to the beaded tube (according to standard protocol) since all microbial samples were diluted in the same 1 × buffer.

Shaking at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL of Wash Buffer (WB) at RT (1000rpm) and mix tubes for 2min

Magnetization to Dynmag-2 for 3min

Removing all s/n

Add 50. mu.L of Lysis Mix (LM) to off-magnet tubes (add 5. mu.L of Polymerase Control (PC) to each PC sample tube)

Carrying out the ETGA reaction: 5min at 1000rpm at 26 ℃ and then 55min at 800rpm

Manual qPCR setup and validation for ETGA (10 μ L reaction)

As a result:

TVC (COL/SAB agar plate)

E cfu/mL values were derived from the highest countable TVC plate

ETGA Ct

Internal Process Control (IPC) Ct

ETGA Δ Ct (average NSC)

Base ofAt the cut-off value of the mean NSC (cfu/mL)

Confirmation of Ct

Positive threshold (Pt) less than or equal to 40Ct

And (3) analysis:

the Magnitor results for the "(+) bead" sample showed that all three microbial species had very strong cell density specific ETGA and confirmation signals, indicating bead specific binding for various microbiomes (GrNeg, GrPos, candida).

Note that the results for Candida may follow a trend towards better cell density, but the liquid culture granulation is quite large and may affect the quality of the serial dilutions

There was some evidence of microbial cell residue in the "(-) bead" control, but this was predicted with only a single wash step.

Example 5: capture of microorganisms from blood by magnetic beads occurs in simple and complex blood lysis buffers, and makes detection of microorganisms comparable to capture by centrifugation

The target is as follows:

two different blood lysis buffers were compared for two different dilution modes for developing a simple and rapid "fast Magnitor" test (no washing step included in the protocol).

And (3) testing conditions are as follows:

2X EBB 1mL 2X EBB +1mL sample

10X EBB 112 μ L10X EBB +1mL sample

2X B-BUF 1mL 2X B-BUF +1mL sample

10X B-BUF 112. mu.L 10X B-BUF +1mL of sample

·10X EBB:500mM Tris-HCl[pH 8.0]+2.5％Tergitol

10X B-BUF 500mM Tris-HCl [ pH8.0] +1.5M sodium chloride + 10% Igepal + 5% sodium deoxycholate + 2.5% Tergitol

Sample setting:

the E.coli o/n liquid culture 1E-3 dilution was added to the blood culture medium (per mL6.25. mu. L o/n: 244. mu. L o/n +39mL blood culture medium) and proliferation was carried out at 37 ℃ and 500rpm for 60 minutes in a shaking incubator

After 2 hours of growth, the sample was generated by adding 1mL of the sample to a 2mL tube containing buffer (and 15 μ L of BioEstapor beads for a magnetic bead sample set (Merck, Cat # BE-M08/0.3)): coli (EC) and no-inclusion control (NSC) samples were run in triplicate for each test condition

100 μ L TVC for NSC and E.coli (plate including dilution of sample to ensure countability)

The scheme is as follows:

the sample is set up as above and immediately subjected to a spinning or bead protocol

Rotation scheme

Centrifuge the sample at 9000Xg for 3min (tube hinge facing outward to trace the pellet)

Removing the supernatant

Add 50. mu.L of LM (about 10-fold pipette mix to resuspend pellet) to sample

Place the sample in a shaking incubator at 900rpm for 5min, and then at 800rpm for 55min (26 ℃ C.)

Centrifuge samples at 17000Xg for 1 min before qpCR setup

Magnetic bead protocol

Place the sample in a shaking incubator at 900rpm for 30min (37 ℃ C.)

Place the sample on DynaMag-2 magnetic stand for 5min, and then remove the supernatant

Add 50. mu.L of LM (about 10-fold pipette mix to resuspend pellet) to sample

Magnetize the sample for 3min before qPCR setup

Manual qPCR settings for ETGA master mix only (10 μ L reaction)

As a result:

cfu calculation

Samples and sample dilutions were plated on COL plates (100. mu.L)

Sample source

ETGA Ct

Summary data

And (3) analysis:

binding of microorganisms by magnetic beads occurs:

omicron simple and complex blood lysis buffers (EBB-Tris-HCl + Tergitol; B-BUF-Tris-HCl + sodium chloride + Igepal + sodium deoxycholate + Tergitol), but the blood-derived test signal varies depending on the blood lysis buffer composition

Sample forms of omicron dilution (2X buffer: 1 part blood lysis buffer to 1 part sample) and concentration (10X buffer: 1 part blood lysis buffer to 9 parts sample)

Furthermore, the microbial detection signal for capturing microbes by magnetic beads is comparable to that by centrifugation.

Example 6: the capture of microorganisms from blood by magnetic beads is not dependent on blood lysis, but when in lysed blood Downstream microbial detection is improved when microbial binding occurs

The target is as follows:

given the recent discovery that multiple bead types/sizes produce similar Magnitor results for microbial serial dilutions and NSCs, it is believed that components within Momentum binding buffers may be mediating/contributing to this observed universal microbial binding profile. To investigate this possibility, E.coli was subjected to a series of dilutions and a standard binding buffer (B-BUF) was compared with a detergent-free B-BUF consisting of Tris-HCl [ pH8.0] + NaCl alone to test whether detergent binding to the microorganism was generally important. Sample sets were prepared for blood culture, pure culture and in 1X binding buffer only to compare results for different sample types.

Preparation:

100mL of 10 Xbinding buffer was freshly prepared:

B-BUF:500mM Tris-HCl [ pH8.0] +1.5M sodium chloride + 10% Igepal + 5% sodium deoxycholate + 2.5% Tergitol

Tris + NaCl: 500mM Tris-HCl [ pH8.0] +1.5M sodium chloride

Estapor beads (Merck, Cat # M1-30/40) were washed 3X 1mL in respective 1 Xbuffer (diluted 10X B-BUF or 10 XTris + NaCl): 40 u L beads heavy suspension in the final volume of 400 u L1X buffer (1% solid content)

The scheme is as follows:

an overnight liquid culture of E.coli was set as a standard in BacTec PLUS aerobic medium, and then 1.88. mu. L o/n was added to 3mL of the culture solution (equivalent to EC 1E-1 dilution added to the culture solution at 6.25. mu.L/mL) the following day (after about 16 hours), and proliferation was performed at 37 ℃ for 2 hours at 500 rpm.

After 2 hours of growth, E.coli precultures were serially diluted (DF10) to EC 1E-6 in either pre-warmed blood culture (BB), neat culture (BO) or 1 Xbuffer (B-BUF or Tris + NaCl).

100 μ L TVC for all E.coli dilutions and NSCs

add 1mL of sample to a 2mL tube containing 112. mu.L of binding buffer (B-BUF or Tris + NaCl: BB and BO samples 10X; and buffer samples 1X) + 15. mu.L of beads (prewashed in the respective buffer)

Shaking at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL of WB at RT (1000rpm) and mix tubes for 2min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 50. mu.L of LM to off-magnet tubes (add 5. mu.L of Polymerase Control (PC) to each PC sample tube)

Manual qPCR setup and validation for ETGA (10 μ L reaction)

And (4) observing results:

as expected, no Tris + NaCl blood lysis was observed

In the absence of detergent, the beads are more granular/agglomerated

As a result:

extrapolation of Cfu/mL values from the highest countable TVC

ETGA Ct

IPC Ct

ETGA Δ Ct (average NSC)

Critical value based on average NSC (cfu/mL)

Confirmation of GrNeR Ct

The positive threshold (Pt) is less than or equal to 40 Ct; false positives

And (3) analysis:

detergents are important to produce good ETGA results in the presence of blood (as indicated by poor ETGA results for "10X Tris + NaCl with BB"), but in the absence of blood lysis, microbial capture/detection remains evident (as evidenced by the results for the "10X Tris + NaCl with BB" sample set).

Good ETGA results in the absence of blood, indicating that detergent as a component of the binding buffer is not necessary for the binding of E.coli to the beads

Good ETGA results in the "10 × Tris + NaCl vs 1 × Tris + NaCl" sample group demonstrate that no biological components in blood and/or culture broth are required for microbial binding.

Interestingly, B-BUF appeared to have a slight inhibition of the ETGA signal in the 10X B-BUF and BO and 1X B-BUF sample groups, but recent work elsewhere showed some inhibition of the assay by sodium deoxycholate, so the observation was not unexpected

The best performance was confirmed in the "10 XTTris + NaCl and 1 XTTris + NaCl" sample groups. All other similar sample groups also yielded similar confirmed GrNeg results.

As can be expected, IPC signals are slightly suppressed by the presence of blood.

These results indicate that neither the detergent nor the biological sample is a microbial binding mediator for E.coli.

Example 7: in the absence of blood, either any pH buffering or salt permeation stabilization occurs Capturing microorganisms by magnetic beads

The target is as follows:

to further investigate the importance of Momentum binding buffers in mediating microbial binding, the effect of pH buffering and salt on binding was investigated in a clean system (i.e. in the absence of any blood or culture broth).

10 buffer preparation:

25mL of buffer were prepared fresh each:

·BUF-1 500mM Tris-HCl[pH7.4]+1.5M NaCl

·BUF-2 500mM Tris-HCl[pH8.0]+1.5M NaCl

·BUF-3 500mM Tris-HCl[pH8.5]+1.5M NaCl

BUF-4 only 500mM Tris-HCl [ pH8.0]

BUF-5 only 1.5M NaCl

BUF-6 Water only

Estapor beads (Merck, Cat M1-30/40) were washed 3 × 1mL in respective 1 × buffer (diluted 10 × buffer): 30 u L beads heavy suspension in the final volume of 300 u L1X buffer (1% solid content)

The scheme is as follows:

coli o/n broth culture set as standards in BacTec PLUS aerobic broth (containing SPS) and nutrient broth (NB without SPS), then 1.88. mu. L o/n was added to 3mL broth (equivalent to EC 1E-1 dilution added to broth at 6.25. mu.L/mL) for each broth type (NB in the morning and PLUS broth in the afternoon) the following day (approximately 16 hours), and 2 hour proliferation incubation was performed at 500rpm at 37 ℃.

For each experiment (NB and PLUS), 1E-1 E.coli precultures were diluted to E.coli 1E-6(DF10) in each 1 Xbuffer (BUF-1 to BUF-6).

100 μ L TVC with separate EC dilution sets performed in relevant media (NB or PLUS media) to prevent plate viability inconsistencies due to different 1 Xbuffers

add 1mL of sample to a 2mL tube containing 112. mu.L of the corresponding 1 Xbuffer + 15. mu.L of beads (prewashed in the corresponding buffer)

Shaking at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL of WB at RT (1000rpm) and mix tubes for 2min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 50. mu.L of LM to the tube from the magnet

Manual qPCR setup and validation for ETGA (10 μ L reaction)

As a result:

NB data set (i.e. no SPS) -morning experiment

All buffers (BUF-1 to 6) NC TVCs ═ 0

Positive threshold (Pt) less than or equal to 40Ct

PLUS data set (i.e. with SPS) -afternoon experiment

All buffers (BUF-1 to 6) NC TVCs ═ 0

Positive threshold (Pt) less than or equal to 40Ct

And (3) analysis:

all buffers (including only water) also showed good capture of E.coli (as shown by similar ETGA and confirmation results) -however, there were some signs of osmotic microbial lysis only in water (BUF-6) at lower cell densities

Coli grown on PLUS and NB produced very similar Magnititor results for all test buffers, indicating that SPS had no significant effect in mediating microbial binding of E.coli

These results indicate that no buffer component is necessary for the binding of E.coli to Estapor (carboxylated) beads

Example 8: the capture of microorganisms from blood by magnetic beads can be performed using a number of different blood lysis methods

The target is as follows:

it was determined whether microbial capture and detection could occur using alternative blood lysis methods.

Preparation:

binding buffers were prepared as follows:

E-BUF ═ 500mM Tris-HCl [ pH8.0] +1.5M sodium chloride + 10% Igepal + 2.5% Tergitol

Urea 83mM Tris-HCl [ pH8.0] +10M urea

Tris + NaCl 500mM Tris-HCl [ pH8.0] +1.5M sodium chloride

BioEstapor beads (Merck, Cat # BE-M08/0.3) were resuspended prior to use.

The scheme is as follows:

staphylococcus aureus o/n liquid culture set as a standard in BacTec PLUS aerobic medium, then the next day (after about 16 hours) 3.0. mu. L o/n was added to 3mL of blood culture (1E-3 dilution) and proliferation was performed at 37 ℃ for 4 hours at 500 rpm.

After 4 hours of growth, the staphylococcus aureus preculture was serially diluted (DF10) to 1E-6 in the preheated blood culture.

100 μ L TVC for all Staphylococcus aureus dilutions and NSCs

Manual sample handling using DynaMag-2 magnet and manual liquid transfer by pipette:

initial setting

For samples using urea, add 0.25mL of sample to a 2mL tube containing 0.75mL of urea +15 μ L of beads

For samples to be frozen, 1mL of sample was added to a 2mL tube, which was then snap frozen on dry ice for 5 min. The samples were thawed at 37 ℃ for 5min and then 112. mu.L Tris + NaCl + 15. mu.L beads were added

For samples using E-BUF or Tris + NaCl, add 1mL of sample to a 2mL tube containing 112. mu.L of binding buffer (E-BUF or Tris + NaCl) + 15. mu.L of beads

Treatment of all samples

Orbital mixing at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL of WB at 37 deg.C (1000rpm) and mix tubes for 3min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 50. mu.L of LM to the tube from the magnet

Manual qPCR setup and validation for ETGA (10 μ L reaction)

And (4) observing results:

blood lysis of thawed frozen samples was observed (see FIG. 1)

Observe blood lysis almost simultaneously with urea

During the treatment of the samples with urea, some beads appeared to be lost

No significant blood lysis was observed for the Tris + NaCl sample group (as expected)

As a result:

extrapolation of Cfu/mL values from the highest countable TVC

ETGA Ct

ETGA ACt (average NSC)

Critical value based on average NSC (cfu/mL)

IPC Ct

Confirmation of GrPos Ct

The positive threshold (Pt) is less than or equal to 40 Ct; false positives

Fig. 1 shows the degree of blood lysis for each sample group: E-BUF, Urea, Tris + NaCl, Freeze (left to right)

And (3) analysis:

capture of microorganisms by magnetic beads and detection of staphylococcus aureus are comparable to other lysis methods and without blood lysis, as determined by confirmation.

However, detection of microorganisms by ETGA is improved to varying degrees by other methods of blood lysis due to the effect on reducing blood-derived ETGA signals.

Example 9: SPS is required in whole blood for optimal bead performance, sample processing and microbiological detection

The target is as follows:

the optimal SPS concentration for the Magnitor rapid test using a 1mL whole blood sample was determined. A secondary objective was to assess the effect of SPS on microbial viability in whole blood as determined by TVS.

And (3) testing conditions are as follows:

for each sample set (E.coli and NSC samples) 2X 5mL whole blood or BacTec PLUS aerobic blood medium was dispensed (1:3 ratio). Then, SPS was added as follows:

then 5u L Escherichia coli 1E-2 preculture was added to each 5mL sample tube, to regenerate standard 1E-5 dilution sample

The scheme is as follows:

adding 1.88. mu.L of purified o/n to 3mL of NB in Nutrient Broth (NB); and incubated at 37 ℃ for 2 hours at 500rpm

After 2 hours, the E.coli preculture was diluted 10-fold in warm NB; and then 5. mu.L of the solution was added to each 5mL sample tube (prepared as shown in test conditions)

The Magnitor test was started immediately and the TVC was performed as detailed below.

pre-load 112 μ L E-BUF (500mM Tris-HCl [ pH8.0] +1.5M sodium chloride + 10% Igepal + 2.5% Tergitol) +15 μ L beads (BioEstapor, Merck, Cat # BE-M08/0.3) into each sample tube, then add 1mL of sample to the sample tube

Orbital mixing at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL of WB at RT (1000rpm) and mix tubes for 3min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 50. mu.L of LM to the tube from the magnet

Manual qPCR setup and validation for ETGA (10 μ L reaction)

As a result:

TVC analysis

100 μ L on COL plates at zero time

The sample contained about 2mL of sample and was placed on a bench (static) at room temperature (20.4 ℃ C.)

TVC was performed at the time points shown in the table: the samples were mixed thoroughly before coating

Magnitor fast test at zero time

ETGA results

Note that: no Ct change to 50Ct for analysis; exclusion of outliers due to significant pellet loss during processing

IPC results

Confirmation of GrNeg results

And (3) analysis:

SPS demonstrates the benefit of providing microbial protection/viability in whole blood based on TVC assay; however, in general, the activity of Escherichia coli in whole blood is not a great problem

Improvement of ETGA andconfirmationSample processing efficiency and microorganism detection performance of both readings

SPS yield 0.06% based on TVC viability, ETGA detection (considering the best results for e.coli and NSC samples Cts), best results for PCR inhibition as indicated by IPC Ct values. It was confirmed that the GrNeg results could also be improved by adding SPS to whole blood, but the exact concentration of SPS was not as critical.

Example 10: various commercially available carboxylated bead products of similar size (-300 nm diameter) were used via magnetic beads from Trapping microorganisms in blood

The target is as follows:

alternative carboxylated magnetic beads of similar size were compared using Momentum's magnet assay

And (3) testing conditions are as follows:

the scheme is as follows:

escherichia coli and Streptococcus pyogenes o/n liquid cultures were set as standards in 3mL of culture and blood culture (BacTec PLUS aerobic) and incubated for 16-20 hours (37 deg.C)

The next day:

e.coli liquid cultures were diluted to 1E-3 in blood culture and then spiked into blood culture (6.25. mu.L per ml of blood culture); and preincubated for 1h 30min (37 ℃ C.)

S. pyogenes liquid cultures were diluted to 1E-1 in blood culture and then spiked into blood culture (6.25. mu.L per ml of blood culture); and preincubated for 2h 30min (37 ℃ C.)

Morning colibacillus experiment

Coli 1E-3 preculture was serially diluted in blood culture to yield 5 dilution points (1E-3 to 1E-7)

Sample set-up by adding 1mL sample to a 2mL tube pre-filled with 15 μ L1% solid beads +112 μ L binding buffer; and subjected to the Magnitor V4.0 test: 5 dilution spots +3 NSCs per bead type (8 sample sets), three bead types were tested on each epMotion 5073m

Streptococcus pyogenes experiment in the afternoon

Streptococcus pyogenes 1E-3 preculture was serially diluted in blood culture to yield 5 dilution points (1E-1 to 1E-5)

Magnitior V4.0 protocol (automatic sample processing of epMotion 5073 m)

Orbital mixing at 37 ℃ for 30min (1000rpm)

Magnetization for 15min

Removal of 1mL s/n

Add 0.82mL of WB to the tube while magnetizing the beads

Removal of 1mL s/n

Add 50. mu.L of LM to the tube while the beads were magnetized

Magnetization was switched off and ETGA reaction was performed: 5min at 1000rpm at 26 ℃ and then 55min at 800rpm

qPCR setup and validation of ETGA (10. mu.L reaction)

As a result:

internal positive threshold (Pt) calculated using NSC (n ═ 6) for each bead type: inc (array, 0.05)

Note that the Pt (5 th%) method will produce a false positive within n ═ 6: highlighted in red font in the following primary results table

Escherichia coli

Confirming that the positive threshold (Pt) is less than or equal to 40 Ct; false positive⁺。

Streptococcus pyogenes

And (4) observing results:

bead B was difficult to resuspend before dilution to 1% solids; and after dilution to 1% solids, visually appears thinner

At the end of the treatment, the sample was placed on a DynaMag-2 magnetic stand and all bead types C-G were magnetized except for: bead a, which appears to have heavy pellets; and beads B with very small beads

And (3) analysis:

all carboxylated magnetic beads tested here showed microbial binding as determined by ETGA and confirmation readings. However, the sensitivity of microbial detection does vary somewhat depending on the level of blood-derived ETGA signal and/or assay inhibition

Example 11: capture of microorganisms from blood by magnetic beads using various bead sizes and functional coatings

The target is as follows:

the microbial capture performance of various commercially available magnetic beads of different sizes and functional coatings were compared using the Momentum' magnet test (ETGA and validation techniques). Two experiments were performed to demonstrate microbial capture for automatic (protocol 1) and manual (protocol 2) sample processing. Importantly, protocol 2 included three bead resuspension washes to more convincingly demonstrate that the ETGA/confirmation signal was specific to bead-bound microbial cells, rather than sample retention (in contrast to protocol 1, protocol 1 included a single bead magnetization wash step).

And (3) testing conditions are as follows:

all beads were washed in 1mL of 1X E-BUF (50mM Tris-HCl [ pH8.0] +150mM sodium chloride + 1% Igepal + 0.25% Tergitol) and resuspended in 1% solids sample set-up in 1X E-BUF (protocol 1 and protocol 2 performed on different dates, respectively):

coli o/n liquid culture set as a standard in 3mL of broth (BacTec PLUS aerobic) and incubated for 16-20 hours (37 ℃)

The following day, E.coli liquid cultures were diluted to 1E-3 in blood culture and subjected to proliferation incubation for 2 hours (37 ℃ @500rpm)

After 2 hours proliferation incubation, E.coli 1E-3 precultures were serially diluted in blood culture to yield three dilution points (EC 1E-6 to 1E-8)

Add 1mL of sample (3 E.coli dilutions and 3 NSC samples: 6 sample groups) to 2mL tubes pre-filled with 112 μ L10X E-BUF (500mM Tris-HCl [ pH8.0] +1.5M sodium chloride + 10% Igepal + 2.5% Tergitol) +15 μ L beads (1% solids), followed by the Magnitor test according to protocol 1 or protocol 2 (see below):

protocol 1 (automated sample processing of epMotion 5073 m):

orbital mixing at 37 ℃ for 30min (1000rpm)

Magnetization for 15min

Removal of 1mL s/n

Add 0.82mL of WB to the tube while magnetizing the beads

Removal of 1mL s/n

Add 50. mu.L of LM to the tube while the beads were magnetized

qPCR setup and validation of ETGA (10. mu.L reaction)

Protocol 2 (manual sample treatment using DynaMag-2 magnet and manual liquid transfer by pipette):

orbital mixing at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL of WB at RT (1000rpm) and mix tubes for 2min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 1mL of WB at RT (1000rpm) and mix tubes for 2min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 1mL of WB at RT (1000rpm) and mix tubes for 2min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 50. mu.L of LM to the tube from the magnet

Manual qPCR setup and validation for ETGA (10 μ L reaction)

As a result:

protocol 1 (automatic sample processing for epMotion 5073 m) -at 20190221

The positive threshold (Pt) is less than or equal to 40 Ct; false positive⁺。

Protocol 2 (manual sample processing using DynaMag-2 magnet and manual liquid transfer by pipette) -at 20190228

And (3) analysis:

these results indicate that a variety of different bead sizes and functional coatings can produce comparable levels of microbial binding, as determined by ETGA and confirmation readings.

Example 12: various microorganisms can be captured from blood using magnetic beads of different sizes and functional coatings (leather) Gram-positive gram-negative candida and gram-negative gram-positive gram-negative gram-positive gram-

The target is as follows:

the microbial capture performance of various commercially available magnetic beads of different sizes and functional coatings were compared using the Momentum' magnet test (ETGA and validation techniques). Coli (bead size and coating I: source experiment: 20190221_ WP7_ beads-comparative-analysis and 20190228_ WP7_ beads-comparative-3-washing-analysis): to expand this previous work, three additional microorganisms (staphylococcus aureus, streptococcus pneumoniae, and candida albicans) were also tested.

And (3) testing conditions are as follows:

all beads were washed in 1mL 1 XTris + NaCl and resuspended to 1% solids in 1 XTris + NaCl

The scheme is as follows:

sample setting:

an overnight liquid culture (o/n) of the microorganism (3 mL of broth inoculated from an agar plate) was set in BacTec PLUS aerobic broth. The next day (approximately 16 hours later), 300. mu.L of Streptococcus pneumoniae and Candida albicans broth cultures were inoculated into 3mL of blood culture (1E-1 dilution), and 3. mu.L of Staphylococcus aureus broth was inoculated into 3mL of blood culture (1E-3 dilution); and was proliferated at 37 ℃ for 2 hours at 500 rpm.

After 2 hours of growth, the microbial precultures were diluted in blood culture (DF10) to produce three dilution points for each microorganism.

100 μ L TVC for each microbial dilution

1mL of sample (3 dilutions and 3 NSC samples per microbial species: 12 sample groups) was added to a 2mL sample tube pre-filled with 15. mu.L of beads (1% solids) and 112. mu. L E-BUF (500mM Tris-HCl [ pH8.0] +1.5M sodium chloride + 10% Igepal + 2.5% Tergitol)

Orbital mixing at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL of WB at RT (1000rpm) and mix tubes for 3min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 50. mu.L of LM to the tube from the magnet

Manual qPCR setup and validation for ETGA (10 μ L reaction)

As a result:

the positive threshold (Pt) is less than or equal to 40 Ct; false positives

And (3) analysis:

these results indicate that various bead sizes and functional coatings produce comparable levels of microbial binding, as determined by ETGA and confirmation readings.

Example 13: magnetic beads of different sizes and functional coatings can be used for capture from simple Tris + NaCl buffers Obtaining various microorganisms (gram-negative, gram-positive and Candida)

The target is as follows:

the microbial capture performance of various commercially available magnetic beads of different sizes and functional coatings were compared using the Momentum' magnet test (ETGA and validation techniques). The experiment was performed using simple buffer (50mM Tris-HCl [ pH8.0] +150mM NaCl) as sample and wash buffer, i.e.no detergent was used before adding the microbial lysis mixture.

And (3) testing conditions are as follows:

all beads were washed in 1mL 1 XTris + NaCl (50mM Tris-HCl [ pH8.0] +150mM NaCl) and resuspended to 1% solids in 1 XTris + NaCl

The scheme is as follows:

sample setting:

an overnight liquid culture (o/n) of the microorganism (3 mL of broth inoculated from an agar plate) was set in BacTec PLUS aerobic broth. The next day (after about 16 hours), 3. mu.L of E.coli and S.aureus broth cultures were inoculated into 3mL of culture broth (1E-3 dilution) and 300. mu.L of C.albicans broth cultures were inoculated into 3mL of culture broth (1E-1 dilution); proliferation was carried out at 37 ℃ and 500rpm for 2 hours.

After 2 hours of growth, the microbial precultures were diluted in 1 × Tris + NaCl buffer (DF10) to produce three dilution points for each microorganism.

100 μ L TVC for each microbial dilution

add 1mL of sample (3 dilutions and 3 NSC samples per microbial species: 12 sample sets) to a 2mL sample tube pre-filled with 15. mu.L beads (1% solids)

Orbital mixing at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL WB (1 × Tris + NaCl) at RT (1000rpm) and mix tubes for 3min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 50. mu.L of LM to the tube from the magnet

Manual qPCR setup and validation for ETGA (10 μ L reaction)

As a result:

the positive threshold (Pt) is less than or equal to 40 Ct; false positives in red

And (3) analysis:

these results indicate that in a clean system (i.e. simple Tris + NaCl buffer instead of biological samples) various different bead sizes and functional surfaces (carboxylated and hydrophobic) all produced comparable levels of microbial binding as determined by ETGA and confirmation readings.

Interestingly, aminated beads (NH2-1.5) produced very poor Magnitor results in the sample type, indicating little/no microbial binding. This observation is different from that in blood culture samples, where aminated beads produce comparable levels of microbial capture to other test beads.

Example 14: magnetic beads of different sizes and functional coatings can be used to capture a variety of microorganisms (gram negative, leather) Lanshi positive and candida)

The target is as follows:

using the Momentum' magnet test (ETGA and validation techniques), the microbial capture performance of various commercially available magnetic beads of different sizes and functional coatings were compared in the absence of blood lysis (i.e., no detergent in the binding buffer)

And (3) testing conditions are as follows:

The scheme is as follows:

sample setting:

an overnight liquid culture (o/n) of the microorganism (3 mL of broth inoculated from an agar plate) was set in BacTec PLUS aerobic broth. The next day (after about 16 hours), 3. mu.L of E.coli and S.aureus broth cultures were inoculated in 3mL of culture (1E-3 dilution) and 300. mu.L of C.albicans broth cultures were inoculated in 3mL of culture (1E-1 dilution); and was proliferated at 37 ℃ for 2 hours at 500 rpm.

100. mu.L of TVC for each microbial dilution

add 1mL of sample (3 dilutions and 3 NSC samples per microbial species: 12 sample groups) to a 2mL sample tube pre-filled with 15. mu.L of beads (1% solids) and 112. mu.L of binding buffer (Tris-HCl + sodium chloride)

Orbital mixing at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL of WB at RT (1000rpm) and mix tubes for 3min

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 50. mu.L of LM to the tube from the magnet

Manual qPCR setup and validation for ETGA (10 μ L reaction)

As a result:

And (3) analysis:

these results indicate that, in the absence of blood lysis, various bead sizes and functional coatings all produce comparable levels of microbial binding from blood, as determined by ETGA and confirmation readings

However, the detection of microorganisms by ETGA was greatly reduced in the absence of blood lysis due to increased NSC ETGA signal compared to previous experiments with blood lysis during microbial binding.

Example 15: capture of microorganisms by magnetic beads occurs in a variety of complex biological sample types

The purpose is as follows:

study of whether microbial Capture Using magnetic beads can be used for other Complex biological fluids besides blood

And (3) testing conditions are as follows:

note that: tris + NaCl: 50mM Tris-HCl [ pH8.0] +150mM sodium chloride

The scheme is as follows:

the E.coli o/n liquid culture was set as a standard in BacTec PLUS aerobic medium, then 3. mu. L o/n was added to 3mL of the culture (E.coli 1E-3) the next day (after about 16 hours) and proliferation incubation was performed at 500rpm for 2 hours at 37 ℃.

After 2 hours of growth, E.coli 1E-3 preculture was diluted to give 5 serial dilution spots (E.coli 1E-6 to 1E-9) in each sample type. 100 μ L of TVC were performed on COL agar plates.

Manual simulation of the magnet using DynaMag-2 magnets and manual liquid delivery:

add 1mL of sample to 2mL of sample tube preloaded with 112. mu.L of binding buffer (500mM Tris-HCl [ pH8.0] +1.5M sodium chloride + 10% Igepal + 2.5% Tergitol + 0.5% sodium deoxycholate) + 15. mu.L beads (BioEstapor beads; Merck # BE-M08/03 (1% solids) — Note that the sample tubes of the Tris + NaCl sample set are not preloaded with 112. mu.L of binding buffer (to avoid the addition of detergents that might inhibit the growth of the microorganisms of the regeneration assay)

Shaking at 37 ℃ for 30min (1000rpm)

Magnetization of DynaMag-2 for 5min

Removing all s/n

Add 1mL WB at RT (1000rpm) and mix tubes for 3 min-Care to add 1mL Tris + NaCl buffer instead of the Tris + NaCl sample group wash buffer to avoid inclusion of detergents that might inhibit the growth of microorganisms in the regeneration assay

Magnetization of Dynmag-2 for 5min

Removing all s/n

Add 50. mu.L LM to tube off magnet-Note, resuspend beads in 100. mu.L Tris + NaCl buffer for regeneration assay

Manual qPCR setup and validation for ETGA (10 μ L reaction)

As a result:

TVCs (100 μ L on COL plate)

Regeneration assay of paired Tris + NaCl sample sets

1.100. mu.l plated/inoculated Tris + NaCl samples- "samples"

2.100. mu.l of supernatant plated/inoculated after the microbial binding step- "after binding"

3.100 μ l of plated/inoculated supernatant after washing step- "after washing"

4.50 μ L resuspended in 100 μ L Tris + NaCl buffer and plated/seeded beads (i.e. 50% material on plate and 50% material seeded into liquid culture) - "beads"

Plates and liquid cultures were incubated overnight at 37 deg.C

Tris + NaCl sample set

Magnitor results:

inc (array, 0.05) was used to calculate a positive threshold (Pt) using NSC (n ═ 3) for each sample type

Positive threshold (Pt) less than or equal to 40Ct

Note that no observable amplification in the Candida channel was used for confirmation

And (3) analysis:

these results indicate that magnetic beads can be used to capture microorganisms from a variety of complex biological sample types, as determined by ETGA and confirmation readings.

The regeneration assay showed that, after binding the magnetic beads, E.coli can be regenerated on agar and liquid cultures, as determined by observable growth of the "bead" sample set.

Example 16: microorganisms can be captured and detected from non-blood samples in the absence of sample lysis

The target is as follows:

microbial capture and detection in non-blood samples of milk and urine using non-lysing binding buffers and non-lysing washing buffers is shown.

Preparation:

10 × Tris + NaCl binding buffer 500mM Tris-HCl [ pH8.0] +1.5M sodium chloride

1/10 dilutions of 1 × Tris + NaCl wash buffer ═ 10 × Tris + NaCl binding buffer

Fresh (human) urine

Semi-skimmed (pasteurized) milk

BioEstapor beads (Merck, Cat # BE-M08/0.3) were resuspended prior to use.

The scheme is as follows:

setting the E.coli, S.aureus, C.albicans and S.pneumoniae o/n liquid cultures as standards in BacTec PLUS aerobic culture

The next day (after about 16 hours), 3. mu.L of E.coli and S.aureus o/n were used to inoculate a fresh 3mL broth culture (1E-3 dilution) and 300. mu.L of Candida albicans and Streptococcus pneumoniae were used to inoculate a fresh 3mL broth culture (1E-1 dilution); proliferation was carried out at 37 ℃ and 500rpm for 2 hours.

After 2 hours of growth, serial dilutions (DF10) of the microbial preculture in pre-warmed fresh urine and fresh milk were made to yield five dilution points: e.coli 1E-5 to 1E-9; staphylococcus aureus 1E-5 to 1E-9; candida albicans 1E-2 to 1E-6; and Streptococcus pneumoniae 1E-2 to 1E-6.

100 μ L TVC for each microbial dilution tested in milk and urine; NSC for milk and urine were plated on three types of agar plates (SAB, COL and CBA)

Add 1mL of sample (five dilutions of each microbial species and four NSC samples: 24 samples of each sample type) to a 2mL sample tube pre-filled with 112. mu.L of binding buffer + 15. mu.L of beads (1% solids) and then initiate the automatic Magnitor test.

Automated sample handling of epMotion 5073 m:

orbital mixing at 37 ℃ for 30min (1000rpm)

Magnetization for 15min

Removal of 1mL s/n

Add 0.82mL WB (1 × Tris + NaCl) to the tube while the beads were magnetized

Removal of 1mL s/n

Add 50. mu.L of LM to the tube while the beads were magnetized

qPCR setup and validation of ETGA (10. mu.L reaction)

As a result:

Cfu/mL values inferred from highest countable TVCs

(Note: urine is a non-sterile solution, therefore, colonies on NSC plates are not unexpected)

(Note: pasteurized milk contains microorganisms, therefore, colonies should be present on NSC plates)

Note that: pasteurized milk contains bacteria; these were shown to be consistent ETGA Ct-22

Confirmation of Ct

Positive threshold (Pt) less than or equal to 40Ct

And (3) analysis:

these results indicate that capture of microorganisms by magnetic beads can replace other samples of blood (particularly urine and milk) in the absence of sample lysis, as determined by ETGA and confirmation readings.

However, the presence of commensal microbes in these sample types (especially milk) affected the background signal level of ETGA and confirmed the readings.

Claims

1. A method of separating microorganisms from non-microbial cells in a sample containing non-microbial cells, the method comprising:

a) incubating the sample with particles to form particle-microorganism complexes, wherein the incubating step is performed in the presence of sodium polyanetholesulfonate and/or an agent that selectively lyses non-microbial cells in the sample while leaving microorganisms present in the sample intact; and

b) separating the particle-microorganism complex from the non-microbial cells.

2. A method of separating microorganisms from non-microbial cells in a sample containing non-microbial cells, the method comprising:

a) incubating the sample with particles to form particle-microorganism complexes; and

b) separating the particle-microorganism complex from the non-microbial cells;

wherein the particles have an outer surface that is not coated with any one of: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein, (iv) a mannose-binding lectin protein.

3. A method of detecting the presence or absence of a microorganism in a sample also comprising non-microbial cells, the method comprising:

a) incubating the sample with particles to form particle-microorganism complexes, wherein the incubating step is performed in the presence of sodium polyanetholesulfonate and/or an agent that selectively lyses non-microbial cells in the sample while leaving microorganisms present in the sample intact;

b) separating the particle-microorganism complex from the non-microbial cells; and

c) detecting the presence or absence of a microorganism in the particle-microorganism complex.

4. A method of detecting the presence or absence of a microorganism in a sample also comprising non-microbial cells, the method comprising:

a) incubating the sample with particles to form particle-microorganism complexes;

c) detecting the presence or absence of a microorganism in the particle-microorganism complex;

5. The method of claim 2 or claim 4, wherein the incubating step is performed in the presence of sodium polyanetholesulfonate and/or an agent that selectively lyses non-microbial cells in the sample while leaving microorganisms present in the sample intact.

6. The method of any one of claims 3 to 5, wherein step (c) comprises: (i) detecting an enzymatic activity of a nucleic acid molecule associated with the microorganism, (ii) directly detecting the microorganism by cell counting or microscopy, (iii) detecting the microorganism after cell culture, (iv) detecting the microorganism by nucleic acid amplification, or (v) detecting the microorganism by nucleic acid sequencing.

7. The method of any one of claims 3 to 5, wherein step (c) comprises the steps of:

i) lysing said microorganisms in said particle-microorganism complex;

ii) incubating the lysate with a nucleic acid molecule that serves as a substrate for the nucleic acid modifying activity of the microorganism; and

iii) specifically determining the presence or absence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying enzyme on the substrate nucleic acid molecule to indicate the presence or absence of the microorganism.

8. The method of claim 7, wherein step (i) comprises adding a cleavage reagent comprising the substrate nucleic acid molecule.

9. The method of claim 7 or claim 8, wherein the nucleic acid modifying enzyme comprises a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I.

10. A method of detecting the presence or absence of a microbial infection in a subject, the method comprising performing the method of any one of claims 3 to 9 on a sample from the subject.

11. The method of any one of the preceding claims, wherein the method further comprises washing the isolated particle-microorganism complex to remove non-microbial cells or lysate.

12. The method of any one of the preceding claims, wherein step b) further comprises removing the non-microbial cells from the particle-microbe complex.

13. The method according to any of the preceding claims, wherein step b) is performed using a magnetic field or centrifugation.

14. A composition, comprising:

a) a particle capable of forming a complex with a microorganism, wherein the particle has an outer surface;

b) sodium polyanetholesulfonate; and

c) at least one agent that selectively lyses non-microbial cells in a sample while leaving intact microorganisms present in the sample.

15. The composition of claim 14, wherein the composition further comprises a sample containing non-microbial cells and possibly microbial cells.

16. A kit for performing the method of any one of claims 1 to 13, the kit comprising:

b) sodium polyanetholesulfonate; and

c) at least one agent that selectively lyses non-microbial cells in the sample while leaving intact microorganisms present in the sample.

17. A kit for performing the method of any one of claims 3 to 13, the kit comprising:

c) particles capable of forming a complex with a microorganism;

d) sodium polyanetholesulfonate;

e) at least one agent that selectively lyses non-microbial cells in the sample while leaving intact microorganisms present in the sample; and

f) detection means for detecting the presence or absence of a microorganism in said particle-microorganism complex, wherein said detection means comprises a nucleic acid molecule (DNA) serving as a substrate for a nucleic acid modifying activity of said microorganism, and wherein said nucleic acid molecule (DNA) is at least partially double-stranded and comprises a uracil residue in the complementary strand.

18. A kit for performing the method of any one of claims 4 to 13, the kit comprising:

a) a particle capable of forming a complex with a microorganism, wherein the particle has an outer surface that is not coated with any one of: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from an apolipoprotein H protein; (iv) mannose-binding lectin protein; and

b) detection means for detecting the presence or absence of a microorganism in said particle-microorganism complex, wherein said detection means comprises a nucleic acid molecule (DNA) serving as a substrate for a nucleic acid modifying activity of said microorganism, and wherein said nucleic acid molecule (DNA) is at least partially double-stranded and comprises a uracil residue in the complementary strand.

19. The kit of claim 18, wherein the kit further comprises reagents that selectively lyse non-microbial cells in the sample while leaving intact microorganisms present in the sample.

20. The composition of claim 14 or claim 15, or the kit of any one of claims 16 to 19, further comprising a buffer and/or sodium chloride.

21. The method of any one of claims 1 to 13, the composition of any one of claims 14, 15 or 20, or the kit of any one of claims 16 to 20, wherein the reagent that selectively lyses non-microbial cells in the sample while leaving microorganisms present in the sample intact is a detergent; optionally wherein the detergent is non-ionic.

22. The method of claim 21, the composition of claim 21 or the kit of claim 21, wherein the detergent is not conjugated to a particle capable of forming a complex with a microorganism.

23. The method of any one of claims 1 to 13, 21 or 22, the composition of any one of claims 14, 15 or 20 to 22, or the kit of any one of claims 16 to 22, wherein the particles have a diameter of between 0.1 μ ι η and 2.0 μ ι η.

24. The method of any one of claims 1 to 13 or 21 to 23, the composition of any one of claims 14, 15 or 20 to 23, or the kit of any one of claims 16 to 23, wherein the particles are magnetic, optionally wherein the particles are superparamagnetic.

25. The method of any one of claims 1 to 13 or 21 to 24, the composition of any one of claims 14, 15 or 20 to 24, or the kit of any one of claims 16 to 24, wherein the outer surface of the particle capable of forming a complex with a microorganism comprises a polymer, optionally wherein the polymer is carbon-based.

26. The method of any one of claims 1 to 13 or 21 to 25, the composition of any one of claims 14, 15 or 20 to 25, or the kit of any one of claims 16 to 25, wherein the outer surface of the particle capable of forming a complex with a microorganism comprises or is coated with any one or more of:

i) a carboxylic acid group;

ii) an amino group;

iii) a hydrophobic group; and

iv) streptavidin.

27. The method of any one of claims 1 to 13 or 21 to 26, the composition of any one of claims 14, 15 or 20 to 26, or the kit of any one of claims 16 to 26, wherein the microorganism is a pathogenic microorganism, optionally wherein the pathogenic microorganism is a pathogenic bacterium or fungus.

28. The method of any one of claims 1 to 13 or 21 to 27, the composition of any one of claims 14, 15 or 20 to 27, or the kit of any one of claims 16 to 27, wherein the non-microbial cells comprise red blood cells and/or white blood cells.

29. The method of any one of claims 1 to 13 or 21 to 28, the composition of any one of claims 14, 15 or 20 to 28, or the kit of any one of claims 16 to 28, wherein the sample comprises blood, urine, saliva or milk, optionally wherein the sample comprises whole blood.